Platelets are anucleated blood cells that participate in a wide range of physiological and pathological functions. Their major role is mediating haemostasis and thrombosis. In addition to these classic functions, platelets have emerged as important players in the innate immune system. In particular, they interact with leukocytes, secrete pro- and anti-inflammatory factors, and express a wide range of inflammatory receptors including Toll-like receptors (TLRs), for example, Toll-like receptor 4 (TLR4). TLR4, which is the most extensively studied TLR in nucleated cells, recognises lipopolysaccharides (LPS) that are compounds of the outer surface of Gram-negative bacteria. Unlike other TLRs, TLR4 is able to signal through both the MyD88-dependent and MyD88-independent signalling pathways. Notably, despite both pathways culminating in the activation of transcription factors, TLR4 has a prominent functional impact on platelet activity, haemostasis, and thrombosis. In this review, we summarise the current knowledge on TLR4 signalling in platelets, critically discuss its impact on platelet function, and highlight the open questions in this area.
In humans, invading pathogens are recognized by Toll-like receptors (TLRs). Upon recognition of lipopolysaccharide (LPS) derived from the cell wall of Gram-negative bacteria, TLR4 dimerizes and can stimulate two different signaling pathways, the proinflammatory, MyD88-dependent pathway and the antiviral, MyD88-independent pathway. The balance between these two pathways is ligand-dependent, and ligand composition determines whether the invading pathogen activates or evades the host immune response. We investigated the dimerization behavior of TLR4 in intact cells in response to different LPS chemotypes through quantitative single-molecule localization microscopy. Quantitative superresolved data showed that TLR4 was monomeric in the absence of its co-receptors MD2 and CD14 in transfected HEK 293 cells. When TLR4 was present together with MD2 and CD14 but in the absence of LPS, 52% of the receptors were monomeric and 48% were dimeric. LPS from or caused the formation of dimeric TLR4 complexes, whereas the antagonistic LPS chemotype from maintained TLR4 in monomeric form at the cell surface. Furthermore, we showed that LPS-dependent dimerization was required for the activation of NF-κB signaling. Together, these data demonstrate ligand-dependent dimerization of TLR4 in the cellular environment, which could pave the way for a molecular understanding of biased signaling downstream of the receptor.
Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.
A distinct feature of the Toll-like receptor 4 (TLR4) is its ability to trigger both MyD88-dependent and MyD88-independent signalling, culminating in activation of pro-inflammatory NF-κB and/or the antiviral IRF3. Although TLR4 agonists (lipopolysaccharides; LPSs) derived from different bacterial species have different endotoxic activity, the impact of LPS chemotype on the downstream signalling is not fully understood. Notably, different TLR4 agonists exhibit antitumoural activity in animal models of glioma, but the underlying molecular mechanisms are largely unknown. Thus, we investigated the impact of LPS chemotype on the signalling events in the human glioma cell line U251. We found that LPS of Escherichia coli origin (LPSEC) leads to NF-κBbiased downstream signalling compared to Salmonella minnesota-derived LPS (LPSSM).Exposure of U251 cells to LPSEC resulted in faster nuclear translocation of the NF-κB subunit p65, higher NF-κB-activity and expression of its targets genes, and higher amount of secreted IL-6 compared to LPSSM. Using super-resolution microscopy we showed that the biased agonism of TLR4 in glioma cells is neither a result of differential regulation of receptor density nor of formation of higher order oligomers. Consistent with previous reports, LPSEC-mediated NF-κB activation led to significantly increased U251 proliferation, whereas LPSSM-induced IRF3 activity negatively influenced their invasiveness. Finally, treatment with methyl-βcyclodextrin (MCD) selectively increased LPSSM-induced nuclear translocation of p65 and NF-κB activity without affecting IRF3.Our data may explain how TLR4 agonists differently affect glioma cell proliferation and migration.
Adult mammalian craniofacial tissues contain limited numbers of post-migratory neural crest-derived stem cells. Similar to their embryonic counterparts, these adult multipotent stem cells can undergo multi-lineage differentiation and are capable of contributing to regeneration of mesodermal and ectodermal cells and tissues in vivo. In the present study, we describe for the first time the presence of Nestin-positive neural crest-derived stem cells (NCSCs) within the ovine hard palate. We show that these cells can be isolated from the palatal tissue and are able to form neurospheres. Ovine NCSCs express the typical neural crest markers Slug and Twist, exhibit high proliferative and migratory activity and are able to differentiate into α smooth muscle cells and β-III-tubulin expressing ectodermal cells. Finally, we demonstrate that oNCSCs are capable of differentiating into osteogenic, adipogenic and chondrogenic cells. Taken together, our results suggest that oNCSCs could be used as model cells to assess the efficacy and safety of autologous NCSC transplantation in a large animal model.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.