Signaling via Toll-like receptor 4 (TLR4) in macrophages constitutes an essential part of the innate immune response to bacterial infections. Detailed and quantified descriptions of TLR4 signal transduction would help to understand and exploit the first-line response of innate immune defense. To date, most mathematical modelling studies were performed on transformed cell lines. However, properties of primary macrophages differ significantly. We therefore studied TLR4-dependent activation of NF-κB transcription factor in bone marrow-derived and peritoneal primary macrophages. We demonstrate that the kinetics of NF-κB phosphorylation and nuclear translocation induced by a wide range of bacterial lipopolysaccharide (LPS) concentrations in primary macrophages is much faster than previously reported for macrophage cell lines. We used a comprehensive combination of experiments and mathematical modeling to understand the mechanisms of this rapid response. We found that elevated basal NF-κB in the nuclei of primary macrophages is a mechanism increasing native macrophage sensitivity and response speed to the infection. Such pre-activated state of macrophages accelerates the NF-κB translocation kinetics in response to low agonist concentrations. These findings enabled us to refine and construct a new model combining both NF-κB phosphorylation and translocation processes and predict the existence of a negative feedback loop inactivating phosphorylated NF-κB.
Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.
In the study, the effect of the TLR4 agonist Immunomax was investigated in vitro and in vivo. In particular, Immunomax was shown to polarize mouse bone marrow macrophages from the M0 and M2 states into the M1 state (ARG1 and iNOS mRNA expression levels were used to identify the mouse M1 and M2 phenotypes). Next, we investigated the prophylactic antiviral effect of Immunomax in both a model of mouse respiratory syncytial virus (RSV) infection and a model of RSV-induced bronchial asthma (BA) exacerbation. In the experiment with RSV-induced BA exacerbation, Immunomax-treated mice were characterized by a significant decrease of the viral load in lung homogenates, an increased amount of M1 macrophages in the lung, a tendency toward Th2-dependent ovalbumin-specific IgG1 antibodies decrease in blood serum, a significant increase in RSV-activated CD4+ T cells secreting IFN (Th1 cells), and a simultaneous significant decrease in the amount of CD4+ cells secreting IL-4 (Th2 cells) in the mouse spleen, which were detected by ELISPOT 1.5 months after experiment. These findings suggest that treatment with the TLR4 agonist Immunomax polarizes the immune response towards antiviral Th1 and may be used for short-term antiviral prophylaxis to prevent acute respiratory viral infections in asthmatics.
BackgroundAgonists of TLR3 and TLR4 are effective immunoadjuvants for different types of vaccines. The mechanisms of their immunostimulatory action differ significantly; these differences are particularly critical for immunization with non-replicating adenovirus vectors (rAds) based vaccines. Unlike traditional vaccines, rAd based vaccines are not designed to capture vaccine antigens from the external environment by antigen presenting cells (APCs), but rather they are targeted to the de novo synthesis of vaccine antigens in APCs transfected with rAd. To date, there is no clear understanding about approaches to improve the efficacy of rAd vaccinations with immunoadjuvants. In this study, we investigated the immunoadjuvant effect of TLR3 and TLR4 agonists on the level of activation of APCs during vaccination with rAds.ResultsWe demonstrated that TLR3 and TLR4 agonists confer different effects on the molecular processes in APCs that determine the efficacy of antigen delivery and activation of antigen-specific CD4+ and CD8+ T cells. APCs activated with agonists of TLR4 were characterized by up-regulated production of target antigen mRNA and protein encoded in rAd, as well as enhanced expression of the co-activation receptors CD80, CD86 and CD40, and pro-inflammatory cytokines TNF-α, IL6 and IL12. These effects of TLR4 agonists have provided a significant increase in the number of antigen-specific CD4+ and CD8+ T cells. TLR3 agonist, on the contrary, inhibited transcription and synthesis of rAd-encoded antigens, but improved expression of CD40 and IFN-β in APCs. The cumulative effect of TLR3 agonist have resulted in only a slight improvement in the activation of antigen-specific T cells. Also, we demonstrated that IFN-β and TNF-α, secreted by APCs in response to TLR3 and TLR4 agonists, respectively, have an opposite effect on the transcription of the targeted gene encoded in rAd. Specifically, IFN-β inhibited, and TNF-α stimulated the expression of target vaccine antigens in APCs.ConclusionsOur data demonstrate that agonists of TLR4 but not TLR3 merit further study as adjuvants for development of vaccines based on recombinant adenoviral vectors.Electronic supplementary materialThe online version of this article (10.1186/s12865-018-0264-x) contains supplementary material, which is available to authorized users.
The article discusses financial management methods and ways to optimize costs. Management of the cost of production of enterprises is a systematic process of formation of costs for the production of all products and the cost of individual products, control over the fulfillment of tasks to reduce the cost of production, identification of reserves for its reduction. Enterprise cost management is a component of the enterprise management system as a whole. Therefore, in general terms, let us dwell on some aspects of enterprise management in order to understand the essence of cost management. Management - the activities of an enterprise aimed at realizing the objectives of the management object, subject to the rational use of available resources.
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