Predicting Novel Features of Toll-Like Receptor 3 Signaling in Macrophages

The Toll-like receptor (TLR) 3 plays a critical role in mammalian innate immune response against viral attacks by recognizing double-stranded RNA (dsRNA) or its synthetic analog polyinosinic-polycytidylic acid (poly (I∶C)). This leads to the activation of MAP kinases and NF-κB which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. To understand the complex interplay of the various intracellular signaling molecules in the regulation of NF-κB and MAP kinases, we developed a computational TLR3 model based upon perturbation-response approach. We curated literature and databases to determine the TLR3 signaling topology specifically for murine macrophages. For initial model creation, we used wildtype temporal activation profiles of MAP kinases and NF-κB and, for model testing, used TRAF6 KO and TRADD KO data. From dynamic simulations we predict i) the existence of missing intermediary steps between extracellular poly (I∶C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation. Our work shows activation dynamics of signaling molecules can be used in conjunction with perturbation-response models to decipher novel signaling features of complicated immune pathways.     

Recovery of Humic-Reducing Bacteria from a Diversity of Environments

To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.     

Toll-Like Receptor Signaling

Toll-like receptors sense pathogen-associated molecular patterns (e.g., lipopolysaccharides) and trigger gene-expression changes that ultimately eradicate the invading microbes.Toll-like receptors (TLRs) are protective immune sentries that sense pathogen-associated molecular patterns (PAMPs) such as unmethylated double-stranded DNA (CpG), single-stranded RNA (ssRNA), lipoproteins, lipopolysaccharide (LPS), and flagellin. In innate immune myeloid cells, TLRs induce the secretion of inflammatory cytokines (Newton and Dixit 2012), thereby engaging lymphocytes to mount an adaptive, antigen-specific immune response (see Fig. 1) that ultimately eradicates the invading microbes (Kawai and Akira 2010).Open in a separate windowFigure 1.TLR signaling (simplified view).Identification of TLR innate immune function began with the discovery that Drosophila mutants in the Toll gene are highly susceptible to fungal infection (Lemaitre et al. 1996). This was soon followed by identification of a human Toll homolog, now known as TLR4 (Medzhitov et al. 1997). To date, 10 TLR family members have been identified in humans, and at least 13 are present in mice. All TLRs consist of an amino-terminal domain, characterized by multiple leucine-rich repeats, and a carboxy-terminal TIR domain that interacts with TIR-containing adaptors. Nucleic acid–sensing TLRs (TLR3, TLR7, TLR8, and TLR9) are localized within endosomal compartments, whereas the other TLRs reside at the plasma membrane (Blasius and Beutler 2010; McGettrick and O’Neill 2010). Trafficking of most TLRs from the endoplasmic reticulum (ER) to either the plasma membrane or endolysosomes is orchestrated by ER-resident proteins such as UNC93B (for TLR3, TLR7, TLR8, and TLR9) and PRAT4A (for TLR1, TLR2, TLR4, TLR7, and TLR9) (Blasius and Beutler 2010). Once in the endolysosomes, TLR3, TLR7, and TLR9 are subject to stepwise proteolytic cleavage, which is required for ligand binding and signaling (Barton and Kagan 2009). For some TLRs, ligand binding is facilitated by coreceptors, including CD14 and MD2.Following ligand engagement, the cytoplasmic TIR domains of the TLRs recruit the signaling adaptors MyD88, TIRAP, TRAM, and/or TRIF (see Fig. 2). Depending on the nature of the adaptor that is used, various kinases (IRAK4, IRAK1, IRAK2, TBK1, and IKKε) and ubiquitin ligases (TRAF6 and pellino 1) are recruited and activated, culminating in the engagement of the NF-κB, type I interferon, p38 MAP kinase (MAPK), and JNK MAPK pathways (Kawai and Akira 2010; Morrison 2012). TRAF6 is modified by K63-linked autoubiquitylation, which enables the recruitment of IκB kinase (IKK) through a ubiquitin-binding domain of the IKKγ (also known as NEMO) subunit. In addition, a ubiquitin-binding domain of TAB2 recognizes ubiquitylated TRAF6, causing activation of the associated TAK1 kinase, which then phosphorylates the IKKβ subunit. Pellino 1 can modify IRAK1 with K63-linked ubiquitin, allowing IRAK1 to recruit IKK directly. TLR4 signaling via the TRIF adaptor protein leads to K63-linked polyubiquitylation of TRAF3, thereby promoting the type I interferon response via interferon regulatory factor (IRFs) (Hacker et al. 2011). Alternatively, TLR4 signaling via MyD88 leads to the activation of TRAF6, which modifies cIAP1 or cIAP2 with K63-linked polyubiquitin (Hacker et al. 2011). The cIAPs are thereby activated to modify TRAF3 with K48-linked polyubiquitin, causing its proteasomal degradation. This allows a TRAF6–TAK1 complex to activate the p38 MAPK pathway and promote inflammatory cytokine production (Hacker et al. 2011). TLR signaling is turned off by various negative regulators: IRAK-M and MyD88 short (MyD88s), which antagonize IRAK1 activation; FADD, which antagonizes MyD88 or IRAKs; SHP1 and SHP2, which dephosphorylate IRAK1 and TBK1, respectively; and A20, which deubiquitylates TRAF6 and IKK (Flannery and Bowie 2010; Kawai and Akira 2010).Open in a separate windowFigure 2.TLR signaling. (Adapted with kind permission of Cell Signaling Technology [http://www.cellsignal.com].)Deregulation of the TLR signaling cascade causes several human diseases. Patients with inherited deficiencies of MyD88, IRAK4, UNC93B1, or TLR3 are susceptible to recurrent bacterial or viral infections (Casanova et al. 2011). Chronic TLR7 and/or TLR9 activation in autoreactive B cells, in contrast, underlies systemic autoimmune diseases (Green and Marshak-Rothstein 2011). Furthermore, oncogenic activating mutations of MyD88 occur frequently in the activated B-cell-like subtype of diffuse large B-cell lymphoma and in other B-cell malignancies (Ngo et al. 2011). Inhibitors of various TLRs or their associated kinases are currently being developed for autoimmune or inflammatory diseases and also hold promise for the treatment of B-cell malignancies with oncogenic MyD88 mutations. Many TLR7 and TLR9 agonists are currently in clinical trials as adjuvants to boost host antitumor responses in cancer patients (Hennessy et al. 2010).     

胰岛素受体介导的信号传递

就胰岛素与其受体结合后, 信号传递的过程及参与信号传递的细胞内信号分子进行了综述.胰岛素作为一种重要激素,参与机体的新陈代谢, 调节细胞的生长分化.其发挥生理功能的第一步是与靶细胞膜上的受体相结合, 激活胰岛素受体的酪氨酸激酶活性, 随之磷酸化细胞内的信号分子, 从而使胰岛素的刺激信号转化为细胞反应.     

Initial Test of the Benchmark Chemical Approach for Predicting Microbial Transformation Rates in Aquatic Environments

Using 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) as a benchmark chemical, we determined relative pseudo-first-order rate coefficients for the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE), methyl parathion, and methyl-3-chlorobenzoate in a diversity of microbial samples, including water, sediment, biofilm, and floating microbial mats collected from a laboratory mesocosm as well as from streams, lakes, and wetlands in Georgia and Florida. The decreasing order of reactivity for relative microbial transformation rates was 2,4-DBE > 2,4-DME > methyl-3-chlorobenzoate > methyl parathion. Half-lives of the chemicals varied about 60-fold depending on the chemical and microbial sample. Relative rate coefficients, however, typically varied only about threefold for field-collected samples. Relative rate coefficients determined with samples from a laboratory mesocosm were consistently low compared with the field sample data. Overall, the data indicated that microbial transformation rates of a chemical can be satisfactorily inferred for a wide variety of microbial habitats—such as water, biofilm, or a sediment—on the basis of its transformation rate relative to that of an appropriate benchmark chemical by using a single type of microbial sample.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.     

Insulin Receptor Signaling in Cones

In humans, age-related macular degeneration and diabetic retinopathy are the most common disorders affecting cones. In retinitis pigmentosa (RP), cone cell death precedes rod cell death. Systemic administration of insulin delays the death of cones in RP mouse models lacking rods. To date there are no studies on the insulin receptor signaling in cones; however, mRNA levels of IR signaling proteins are significantly higher in cone-dominant neural retina leucine zipper (Nrl) knock-out mouse retinas compared with wild type rod-dominant retinas. We previously reported that conditional deletion of the p85α subunit of phosphoinositide 3-kinase (PI3K) in cones resulted in age-related cone degeneration, and the phenotype was not rescued by healthy rods, raising the question of why cones are not protected by the rod-derived cone survival factors. Interestingly, systemic administration of insulin has been shown to delay the death of cones in mouse models of RP lacking rods. These observations led to the hypothesis that cones may have their own endogenous neuroprotective pathway, or rod-derived cone survival factors may be signaled through cone PI3K. To test this hypothesis we generated p85α^−/−/Nrl^−/− double knock-out mice and also rhodopsin mutant mice lacking p85α and examined the effect of the p85α subunit of PI3K on cone survival. We found that the rate of cone degeneration is significantly faster in both of these models compared with respective mice with competent p85α. These studies suggest that cones may have their own endogenous PI3K-mediated neuroprotective pathway in addition to the cone viability survival signals derived from rods.     

Chemical Characterization of Exopolysaccharides from Antarctic Marine Bacteria

Exopolysaccharides (EPS) may have an important role in the Antarctic marine environment, possibly acting as ligands for trace metal nutrients such as iron or providing cryoprotection for growth at low temperature and high salinity. Ten bacterial strains, isolated from Southern Ocean particulate material or from sea ice, were characterized. Whole cell fatty acid profiles and 16S rRNA gene sequences showed that the isolates included representatives of the genera Pseudoalteromonas, Shewanella, Polaribacter, and Flavobacterium as well as one strain, which constituted a new bacterial genus in the family Flavobacteriaceae. The isolates are, therefore, members of the “Gammaproteobacteria” and Cytophaga-Flexibacter-Bacteroides, the taxonomic groups that have been shown to dominate polar sea ice and seawater microbial communities. Exopolysaccharides produced by Antarctic isolates were characterized. Chemical composition and molecular weight data revealed that these EPS were very diverse, even among six closely related Pseudoalteromonas isolates. Most of the EPS contained charged uronic acid residues; several also contained sulfate groups. Some strain produced unusually large polymers (molecular weight up to 5.7 MDa) including one strain in which EPS synthesis is stimulated by low temperature. This study represents a first step in the understanding of the role of bacterial EPS in the Antarctic marine environment.     

Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments

Both magnetic collection and “race track” purification techniques were highly effective for selective enrichment of magnetotactic bacteria (MTB) from complex communities, as suggested by amplified ribosomal DNA restriction analysis and denaturing gradient gel electrophoresis combined with sequence analysis of 16S rRNA genes. Using these purification methods, the occurrence and diversity of MTB in microcosms from various marine and freshwater environments were assayed by using a combined microscopic, molecular, and cultivation approach. Most microcosms were dominated by magnetotactic cocci. Consistently, the majority of retrieved 16S RNA sequences were affiliated with a distinct cluster in the Alphaproteobacteria. Within this lineage the levels of sequence divergence were <1 to 11%, indicating genus-level diversity between magnetotactic cocci from various microcosms, as well as between MTB from different stages of succession of the same microcosms. The community composition in microscosms underwent drastic succession during incubation, and significant heterogeneities were observed between microcosms from the same environmental sources. A novel magnetotactic rod (MHB-1) was detected in a sediment sample from a lake in northern Germany by fluorescence in situ hybridization. MHB-1 falls into the Nitrospira phylum, displaying 91% 16S rRNA sequence similarity to “Magnetobacterium bavaricum.” In extensive cultivation attempts, we failed to isolate MHB-1, as well as most other MTB present in our samples. However, although magnetotactic spirilla were not frequently observed in the enrichments, 10 novel isolates of the genus Magnetospirillum which had not routinely been isolated in pure culture before were obtained.     

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α and RC-3095 (10 ng/mL). Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis, GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal–related kinase (ERK)-1/2, Jun NH₂-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-κB and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-α. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP, which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling.     

How Location Governs Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are a family of innate immune system receptors responsible for recognizing conserved pathogen-associated molecular patterns (PAMPs). PAMP binding to TLRs initiates intracellular signaling pathways that lead to the upregulation of a variety of costimulatory molecules and the synthesis and secretion of various cytokines and interferons by cells of the innate immune system. TLR-induced innate immune responses are a prerequisite for the generation of most adaptive immune responses, and in the case of B cells, TLRs directly regulate signaling from the antigen-specific B-cell receptor. The outcome of TLR signaling is determined, in part, by the cells in which they are expressed and by the selective use of signaling adaptors. Recent studies suggest that, in addition, both the ligand recognition by TLRs and the functional outcome of ligand binding are governed by the subcellular location of the TLRs and their signaling adaptors. In this review we describe what is known about the intracellular trafficking and compartmentalization of TLRs in innate system's dendritic cells and macrophages and in adaptive system's B cells, highlighting how location regulates TLR function.     

Chemical Signaling in Ciliates

ABSTRACT. For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated gamones identified in culture filtrates of a Blepharisma species. However, the characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone-receptor interactions and thus, provide important clues into pheromone mechanisms of action.     

Cocaine-Induced Changes in NMDA Receptor Signaling

Addictive states are often thought to rely on lasting modification of signaling at relevant synapses. A long-standing theory posits that activity at N-methyl-d-aspartate receptors (NMDARs) is a critical component of long-term synaptic plasticity in many brain areas. Indeed, NMDAR signaling has been found to play a role in the etiology of addictive states, in particular, following cocaine exposure. However, no consensus is apparent with respect to the specific effects of cocaine exposure on NMDARs. Part of the difficulty lies in the fact that NMDARs interact extensively with multiple membrane proteins and intracellular signaling cascades. This allows for highly heterogeneous patterns of NMDAR regulation by cocaine in distinct brain regions and at distinct synapses. The picture is further complicated by findings that cocaine effects on NMDARs are sensitive to the behavioral history of cocaine exposure such as the mode of cocaine administration. This review provides a summary of evidence for cocaine-induced changes in NMDAR expression, cocaine-induced alterations in NMDAR function, and cocaine effects on NMDAR control of intracellular signaling cascades.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.