RESUMO
Esophageal adenocarcinoma (EAC) develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor (TLR) pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171 (14.6%, PathScan p = 8.7x10-5) tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett's esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149 (22.1%, PathScan p = 0.05) tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database (10,318 samples), including uterine endometrioid carcinoma (188/320, 58.8%), cutaneous melanoma (377/988, 38.2%), colorectal adenocarcinoma (402/1519, 26.5%), and stomach adenocarcinoma (151/579, 26.1%). TLR4 was the most frequently mutated gene with eleven mutations in 10/171 (5.8%) of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients.
Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Mutação , Receptor 4 Toll-Like/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Células HEK293 , Humanos , Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Receptor 4 Toll-Like/metabolismoRESUMO
As we learn more about the biology of the Toll-like receptors (TLRs), a wide range of molecules that can activate this fascinating family of pattern recognition receptors emerges. In addition to conserved pathogenic components, endogenous danger signals created upon tissue damage are also sensed by TLRs. Detection of these types of stimuli results in TLR mediated inflammation that is vital to fight pathogenic invasion and drive tissue repair. Aberrant activation of TLRs by pathogenic and endogenous ligands has also been linked with the pathogenesis of an increasing number of infectious and autoimmune diseases, respectively. Most recently, allergen activation of TLRs has also been described, creating a third broad class of TLR stimulus that has helped to shed light on the pathogenesis of allergic disease. To date, microbial activation of TLRs remains best characterized. Each member of the TLR family senses a specific subset of pathogenic ligands, pathogen associated molecular patterns (PAMPS), and a wealth of structural and biochemical data continues to reveal the molecular mechanisms of TLR activation by PAMPs, and to demonstrate how receptor specificity is achieved. In contrast, the mechanisms by which endogenous molecules and allergens activate TLRs remain much more mysterious. Here, we provide an overview of our current knowledge of how very diverse stimuli activate the same TLRs and the structural basis of these modes of immunity.
Assuntos
Modelos Biológicos , Transdução de Sinais , Receptores Toll-Like/agonistas , Alérgenos/metabolismo , Animais , Autoimunidade , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Humanos , Imunidade Inata , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Conformação Proteica , Isoformas de Proteínas , Receptores Toll-Like/química , Receptores Toll-Like/metabolismoRESUMO
DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.
Assuntos
Lipopolissacarídeos/química , Receptor 4 Toll-Like/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Cavalos , Humanos , Metabolismo dos Lipídeos , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/metabolismo , Antígeno 96 de Linfócito/fisiologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Recombinantes de Fusão , Transdução de Sinais , Especificidade da Espécie , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/fisiologiaRESUMO
Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS.
Assuntos
Antígenos de Superfície/biossíntese , Meios de Cultura , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/biossíntese , Antígeno 96 de Linfócito/isolamento & purificação , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Cromatografia em Gel , Clonagem Molecular , Dissulfetos/química , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Cavalos , Humanos , Ligantes , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/genética , Fenótipo , Pichia/química , Pichia/genética , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate murine antigen-presenting cells in vitro. This agonistic effect was further confirmed in silico using a prediction model based on crystal structures. Also, lipidoids formulated as lipoplexes or as stable nucleic acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation design.
RESUMO
Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and â¼12 fold higher IFN-ß gene expression levels compared to wild-type subjects (P<0.05; MWU test) and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.