Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3.
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ABSTRACT:
SUBMITTER: Ranjith-Kumar CT
PROVIDER: S-EPMC2447120 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.
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Project description:Up to a third of the world's population is infected with Toxoplasma gondii. Natural infection in humans can be life threatening during pregnancy and in immunocompromised individuals. Toll-like receptor (TLR) 11 is the mouse innate sensor that recognizes T. gondii profilin; however, in humans the TLR11 gene leads to transcription of no functional protein. Herein, by using a multiple sequence alignment phylogenetic analysis program between human and mouse species, we found that human TLR5 seems to be the evolutionarily closest member of the TLR gene family to mouse tlr11. We therefore asked whether human TLR5 could mediate IL-6, IL-8 and IL-12p70 production in response to the T. gondii profilin. We found that this was the case both in human cell lines as well as peripheral blood monocytes. Moreover, TLR5 neutralization and gene silencing mediated specific ablation of cytokine production after profilin exposure. Finally, peripheral blood monocytes carrying the TLR5 R392X mutation failed to produce cytokines in response to stimulation with profilin. Taken together, the results presented herein reveal a previously unappreciated cross-recognition of a relevant human pathogen-derived pathogen-associated molecular pattern.
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Project description:Toll-like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase family (IRAK-1, 2, M and 4). IRAK-1 and IRAK-2, known to form Myddosomes with MyD88-IRAK-4, mediate TLR7-induced TAK1-dependent NF?B activation. IRAK-M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK-M was also able to interact with MyD88-IRAK-4 to form IRAK-M Myddosome to mediate TLR7-induced MEKK3-dependent second wave NF?B activation, which is uncoupled from post-transcriptional regulation. As a result, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and I?B?), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NF?B activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.
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Project description:ObjectivesCatecholaminergic vasopressors are the cornerstone of circulatory shock management. Nevertheless, catecholamines have problematic side effects, arousing a growing interest in noncatecholaminergic agents such as vasopressin or angiotensin-II. However, their respective effects on sepsis-associated microvascular endothelial dysfunction such as permeability or inflammation remain elusive. We investigated the role of catecholamines and other vasopressors on Toll-like receptor agonists-induced microvascular endothelial permeability and inflammation.SettingUniversity research laboratory/cell research.SubjectsHuman pulmonary microvascular endothelial cells from multiple donors.InterventionConfluent monolayers of human pulmonary microvascular endothelial cells were treated with Toll-like receptor agonists (lipopolysaccharide, Poly[I:C], or tripalmitoyl-S-glyceryl cysteine) in the presence or absence of epinephrine, norepinephrine, vasopressin, and angiotensin-II. Permeability was inferred from transendothelial resistance, measured using electrical cell impedance sensing, where decreased transendothelial resistance is consistent with increased permeability. Cell-cell junction molecule expression was assessed via immunofluorescence microscopy and flow cytometry. We quantified cytokines in supernatants of Toll-like receptor agonist-treated human pulmonary microvascular endothelial cells.Measurements and main resultsEpinephrine and norepinephrine both ameliorate lipopolysaccharide, polyinosinic:polycytidylic acid, or tripalmitoyl-S-glyceryl cysteine-induced reductions in transendothelial resistance, a surrogate for endothelial permeability. In contrast, the noncatecholaminergic agents, vasopressin, and angiotensin-II did not affect Toll-like receptor agonists-induced reductions in transendothelial resistance. β1- and β2-adrenergic receptor antagonists reduced the effects of the catecholamines on transendothelial resistance, whereas α-adrenergic receptor antagonists did not. We observed that epinephrine and norepinephrine induced actin cytoskeletal rearrangement and normalized the membrane expression of proteins involved with adherens-junctions (vascular endothelial-cadherin) and tight-junctions (zona occludens-1). Despite having a substantial effect on endothelial permeability, epinephrine and norepinephrine did not affect human pulmonary microvascular endothelial cell survival or production of interleukin-8, interleukin-6, or monocyte chemoattractant protein-1 (CCL-2) induced by Toll-like receptor agonists, suggesting that these functions are regulated separately from permeability.ConclusionsOur findings demonstrate that treatment with epinephrine or norepinephrine strongly reduces endothelial permeability induced by agonists of multiple Toll-like receptors (Toll-like receptor-2, Toll-like receptor-3, Toll-like receptor-4) in vitro. Our studies suggest that both β1- and β2-adrenergic receptors mediate the stabilizing effects of epinephrine and norepinephrine on the endothelial barrier.
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Project description:Neonates rely on their innate immune system, and neutrophils in particular, to recognize and combat life-threatening bacterial infections. Pretreatment with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 agonist, improves survival to polymicrobial sepsis in neonatal mice by enhancing neutrophil recruitment. To understand the response of human neonatal neutrophils to TLR4 stimulation, ex vivo spontaneous neutrophil migration, neutrophil transcriptomics, and cytokine production in the presence and absence of LPS were measured directly from whole blood of adults, term neonates, and preterm neonates. Spontaneous neutrophil migration was measured on novel microfluidic devices with time-lapse imaging for 10 h. Genome-wide neutrophil transcriptomics and plasma cytokine concentrations were also determined. Preterm neonates had significantly fewer spontaneously migrating neutrophils at baseline, and both term and preterm neonates had decreased neutrophil velocity, compared to adults. In the presence of LPS stimulation, the number of spontaneously migrating neutrophils was reduced in preterm neonates compared to term neonates and adults. Neutrophil velocity was not significantly different among groups with LPS stimulation. Preterm neonates upregulated expression of genes associated with the recruitment and response of neutrophils following LPS stimulation, but failed to upregulate the expression of genes associated with antimicrobial and antiviral responses. Plasma levels of IL-1β, IL-6, IL-8, MIP-1α, and TNF-α increased in response to LPS stimulation in all groups, but IL-10 was increased only in term and preterm neonates. In conclusion, age-specific changes in spontaneous neutrophil migration counts are not affected by LPS despite changes in gene expression and cytokine production. KEY MESSAGES:Preterm neonates have reduced spontaneous neutrophil migration compared to term neonates and adults in the absence and presence of TLR4 stimulation. Preterm and term neonates have reduced neutrophil velocities compared to adults in the absence of TLR4 stimulation but no difference in the presence of TLR4 stimulation. Unique transcriptomic response to TLR4 stimulation is observed in neutrophils from preterm neonates, term neonates, and adults. TLR4 stimulation produces an age-specific cytokine response.
| S-EPMC6091213 | biostudies-literature
Project description:Toll-like receptors (TLRs) are pivotal in innate immunity and inflammation. Here we show that genetic deficiency in Peli1, an E3 ubiquitin ligase, attenuated the induction of proinflammatory cytokines by ligands of TLR3 and TLR4 and rendered mice resistant to septic shock. Peli1 was required for TLR3-induced activation of IkappaB kinase (IKK) and its 'downstream' target, transcription factor NF-kappaB, but was dispensable for IKK-NF-kappaB activation induced by several other TLRs and the interleukin 1 (IL-1) receptor. Notably, Peli1 bound to and ubiquitinated RIP1, a signaling molecule that mediates IKK activation induced by the TLR3 and TLR4 adaptor TRIF. Our findings suggest that Peli1 is a ubiquitin ligase needed for the transmission of TRIF-dependent TLR signals.
| S-EPMC2748822 | biostudies-literature
Project description:Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release. TLR2 KO mice were backcrossed with C57BL/6 animals for a minimum of eight generations prior to use in these studies. Age- and sex-matched C57BL/6 mice were used as wild type (WT) controls. Primary mixed glial cultures were prepared from the cerebral corticies of neonatal mice (2-4 days of age) and microglia were harvested using a differential shaking technique with a purity of >98%. A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was used to stimulate the microglia isolates. Bacterial strains were heat-inactivated and used to stimulate microglia at 107 colony forming units (cfu)/well for 6 and 12 hours time points. Three replicates of each mouse type (WT, TLR2 KO) at both time points 6 and 12 hours were used for the microarray experiments. Data was only usable for 2 replicates of the KO-12 hr group.
2014-12-22 | E-GEOD-24935 | biostudies-arrayexpress
Project description:Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.
| S-EPMC5458134 | biostudies-literature
Project description:Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3? and 14-3-3? proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3? and 14-3-3? in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNF?, and IFN-? production. We also show that 14-3-3? and 14-3-3? impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-?B and IFN-? reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam(3)CSK(4), LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3? and 14-3-3? also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3? and 14-3-3? play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.
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Project description:BackgroundSingle-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure.ResultsThe minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with [Formula: see text] of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only [Formula: see text] of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5-10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions.ConclusionsIn general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.
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