Transcriptional response of macrophages to combined stimulation of NOD-like and Toll-like receptors (report 2)
Introduction. Combinations of NOD-like and Toll-like receptor agonists are potent activators of macrophages. However, there is lack of detailed characteristics of transcriptional alterations occurring in macrophages stimulated by these agonists combinations.
Aim – to reveal and compare groups of genes characterized by increased and reduced expression upon combined stimulation of human macrophages with NOD1 and TLR4 agonists in vitro.
Material and methods. Macrophages generated from healthy donor monocytes were stimulated with NOD1 and TLR4 agonists separately or simultaneously during 1 or 4 hours. Transcriptomes were analysed using high-throughput RNA sequencing (RNA-seq) and bioinformatics.
Results. In macrophages stimulated with NOD1 + TLR4 agonist combination, we identified two groups of genes (> 500 genes each) characterized by either augmented or repressed mRNA expression. The inducible group was strongly enriched with genes encoding key inflammatory mediators, except for typical markers of M1 activation. The repressible group was enriched with genes coding for cell cycle regulators. Using bioinformatics approaches, we identified potential mechanisms that may underlie transcriptional reprogramming of macrophages in these activation conditions.
Conclusions. Combination of NOD1 and TLR4 agonists induces transcriptional reprogramming of macrophages compatible with innate type of activation.
Suppression of respiratory syncytial virus infection in vitro by small interfering RNAs complexed with bifunctional peptide carrier
Introduction. Respiratory syncytial virus (RSV) is one of the most common pathogens affecting the upper and lower respiratory tract. One of the promising approaches to the creation of antiviral drugs is the use of small interfering RNA (siRNA) molecules that can specifically block the expression of virus genes. Another approach is the use of natural and synthetic peptides as antiviral agents. Notably, peptides can possess several biological properties. For example, we showed that the cationic dendrimeric peptide LTP not only exhibited antiviral properties against RSV, but was also able to transport nucleic acids into epithelial cells.
The aims of this study were to create a complex of bifunctional peptide LTP and siRNA molecules directed against vital RSV genes and to study its antiviral properties in vitro.
Material and methods. The optimal ratio of the component in the siRNA/peptide complex was determined by eukaryotic cell transfection. The toxic effect of the complex on cells was studied by the standard in vitro method (MTT-test). The antiviral properties of the complex were evaluated in the in vitro model of RSV infection. Viral load was assessed by titration on a monolayer of sensitive cells and by quantitative PCR analysis.
Results. We have shown that the optimal ratio of components in the siRNA/LTP complex is 1/12.5 by weight. With this ratio siRNA and peptide molecules form nanostructures with a diameter of 714 ± 125 nm, which have a positive charge of 16.9 ± 6.71 mV. Such physicochemical characteristics allow the complex to penetrate into cells. It was demonstrated that the complex and its individual components are non-cytotoxic in concentrations that have an antiviral effect. We showed that the siRNA/peptide complex provided a more pronounced suppression of virus replication compared to the free peptide. The enhanced antiviral effect of the complex is achieved due to the fact that the peptide has its own antiviral properties and additionally transports siRNA molecules blocking the replication of the virus genome into infected cells.
Conclusion. The complex of bifunctional LTP peptide and siRNA molecules directed against the vital RSV genes provides a more pronounced antiviral effect compared to peptide alone.
Study of the antigenic specificity of T-cell immune reactions in response to immunization of laboratory mice with a recombinant adenoviral vector encoding the Spike-protein of SARS-CoV-2
Introduction. Recombinant adenoviral vectors become the leading technological platform in the development and production of modern vaccines. In Russia and other countries of the world, vaccines based on recombinant adenoviruses have been designed and registered against the Ebola virus and SARS-CoV-2 coronavirus infections. Clinical trials of vaccines against influenza, Marburg virus, human papillomaviruses are ongoing. The target antigen encoded in the DNA of the adenoviral vector is expressed in the body of the vaccine recipient, and adaptive immune responses develop against this target antigen protein. The vector particle is viral and contains dozens of viral antigens. Therefore, along with immune responses to the encoded target antigen, immune responses to the antigens of the vector itself can develop in the body of the vaccinated.
The aim of this work is to investigate the intensity and quality of two immune responses that are different in their antigenic specificity, i.e. directed against the target coronavirus antigen and adenovirus vector antigens.
Material and methods. In C57BL/6 mice, the intensity of immune responses of CD4 and CD8 T cells was studied in response to immunization with a recombinant adenoviral vector encoding the SARS-CoV-2 S-protein (Ad5-S). 2 and 3 months after immunization, the number and antigenic specificity of CD4 and CD8 T memory cells were determined in the spleen of mice. The T cell response was induced in vitro in co-culture with antigen-presenting dendritic cells. Purified CD4 and CD8 T cell populations were obtained by sorting on a BD FACS Aria II laser flow sorter machine. Antigen-presenting cells were transduced with the Ad5-S adenoviral vector encoding the SARS-CoV-2 S-protein or a control recombinant adenoviral vector without a target insert (Ad5-0). The T cells response was determined by ELISPOT according to the number of cells secreting IFN-γ. In some experiments, to reactivate CD4 T cells, antigen-presenting dendritic cells were loaded with a recombinant RBD-fragment of the SARS-CoV-2 S-protein. To enhance the T cell response, antigen presenting dendritic cells were stimulated with a TLR4 agonist.
Results. A single intranasal immunization with the Ad5-S vector at a dose of 108 PFU induced strong systemic T-cell immune responses in C57BL/6 mice. Two months after immunization, about 100–200 thousand antigen-reactive memory T-cells were found in the spleen of mice, secreting IFN-γ when reactivated in vitro by dendritic cells presenting the target SARS-CoV-2 S-antigen. Most antigen-reactive CD8-T memory cells were specific for the SARS-CoV-2 S-antigen. The contents of such cells after immunization with the Ad5-S vector exceeds 1 % of all CD8-T cells. The number of antigen-reactive CD8-T memory cells specific to the adenoviral antigens of the vector was approximately 3-fold lower than the number of antigen-reactive CD8-T memory cells specific to the target S-antigen. The intensity of the immune response of CD4-T cells to immunization with the Ad5-S vector was comparable to the intensity of the immune response of CD8-T cells. The vast majority of antigen-reactive CD4-T memory cells were specific for adenovirus vector antigens. These CD4-T cells secreted IFN-γ in response to in vitro restimulation by dendritic cells transduced with the recombinant adenoviral Ad5-0 vector without a targeted insert. The number of CD4-T cells responding to restimulation by dendritic cells loaded with recombinant RBD was vanishingly small.
An intensity of the response of CD8-T cells specific to the target S-antigen can be increased by elevating of the Ad5-S vector dose during transduction of antigen-presenting dendritic cells, as well as by stimulation of antigen-presenting dendritic cells with a TLR4 agonist.
Possible mechanisms of cooperative interaction between CD8-T cells specific for the target coronavirus S-antigen and CD4-T cells specific for adenovirus vector antigens have been proposed. Possible ways to enhance the response of CD4-T cells to the target S-antigen are considered.
Conclusion. Immunization with a recombinant adenoviral vector encoding the coronavirus S-antigen induces strong CD8- and CD4-T cell immune responses with the formation of a massive pool of antigen-reactive memory T cells. CD8- and CD4-T cells responding to immunization with the Ad5-S vector differ in their antigen specificity. Memory CD8-T cells are generally specific for the target coronavirus S-antigen. Memory CD4-T cells are specific to adenovirus vector antigens.
Production of cytokines by in vitro stimulated naive B lymphocytes and memory B cells
Introduction. Much attention is paid to the description of the role of individual cytokines in the induction of the differentiation of various subpopulations of B cells into plasmablasts and plasma cells. In contrast, little is known about which cytokines are produced by B-lymphocytes themselves and their subpopulations.
The aim of the study was to determine the cytokine profile of human B-lymphocytes during in vitro stimulation. The objectives of the study also included a comparative study of the spectrum of cytokines secreted by naive B-lymphocytes, as well as memory B-cells with switched and unswitched Ig synthesis.
Material and methods. Subpopulations of naive B-lymphocytes, as well as memory B-cells with switched (IgG+CD27+) and non-switched (IgM+CD27+) Ig synthesis were isolated using a flow cytometer. The isolated B lymphocytes were stimulated in vitro in the presence of feeder cells carrying the CD40L molecule and exogenous IL-21. Supernatants collected from stimulated B cells were analyzed for the presence of cytokines. The study was screening in nature. In order to cover the widest possible panel of lymphokines, a high-performance method of multiplex analysis was used in the work. The tested panel included the following lymphokines: EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12P40, IL-12P70, IL-13, IL-15, IL-17A, IL-1RA, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα, TNFβ, VEGF, FGF-2, TGF-α, Flt-3L, Fractalkine, GRO, MCP-3, MDC, PDGF-AA, PDGF-AB/BB and IL-9.
Results. Three subpopulations of stimulated B lymphocytes were obtained and functionally characterized: naive B-lymphocytes, memory B cells with switched (IgG+CD27+) and unswitched (IgM+CD27+) Ig synthesis. Stimulated B lymphocytes were characterized by active proliferation, acquired the plasmablast phenotype and secreted Ig. B lymphocytes stimulated in vitro in the IL-21/CD40L system produced a wide range of cytokines. IP-10, MDC and MCP-1 were secreted to the greatest extent. The secretion of IL-17A, IL-12P70, TGFα, IFN-γ, IL-3, IL-5 and IL-1β was below the level of detection. Naive B cells secreted the cytokines IL-10, MCP-3, MDC, IL-1α, MCP-1, TNFα and TNFβ more actively than memory B cells. For the cytokines IL-10, MDC, MCP-1, TNFα, and TNFβ, a significant difference was observed for both subpopulations of memory B cells. For MCP-3, a significant difference was observed only for memory B cells with switched Ig synthesis, and for IL-1α only when compared with memory B cells with unswitched Ig synthesis.
Conclusion. Cytokine profiles of activated B lymphocytes and their subpopulations were determined. The obtained results show that the production of cytokines by B cells is largely dependent on the activation and differentiation of B lymphocytes.
Synthetic siRNA molecules suppress Il4 and Il13 gene expression and reduce allergic inflammation and remodeling of upper airways in mouse model
Introduction. Inflammation of the upper airways is usually associated with allergic rhinitis (AR), chronic rhinosinusitis with nasal polyps (CRSwNP) etc. AR is one of the most common inflammatory diseases. Its pathogenesis includes increased production of allergen-specific IgE antibodies, infiltration of the nasal mucosa with inflammatory cells, and remodeling of the respiratory tract. It has been shown that Th2-cytokines (IL-4 and IL-13) play a key role in this pathology, which makes them promising targets for therapy. Drugs based on monoclonal antibodies targeted to these cytokines have been developed and demonstrated clinical efficacy, while its broad use is limited by high cost. Currently, new technologies for regulation of gene expression are developed. For example, small interfering RNA (siRNA) molecules can suppress the expression of genes encoding pro-inflammatory cytokines.
The aim of this study was to develop siRNAs suppressing the expression of Il4 and Il13 genes and to reveal their biological effects on the inflammation of the upper respiratory tract in a mouse model.
Material and methods. The design of siRNA molecules was carried out using specialized software. Screening of siRNA biological activity was performed by in vitro experiments. The production of IL-4 and IL-13 was evaluated by ELISA and the expression of the corresponding genes by real-time polymerase chain reaction (RT-PCR). Induction of upper respiratory tract inflammation in mice was performed by subcutaneous and intranasal administration of ovalbumin chicken egg allergen (OVA). Experimental therapy was carried out by intranasal administration of siRNA molecules in complex with the LTP carrier peptide. A corticosteroid drug, budesonide, was used as a positive control. Nasal hyperreactivity was evaluated. Levels of allergen-specific IgE, IgG1 and IgG2a antibodies in mice blood sera, as well as levels of IL-4 and IL-13 production by submandibular lymph nodes were determined by ELISA. Infiltration of the nasal mucosa by proinflammatory cells was assessed by histological methods.
Results. Intranasal administration of siRNAs targeted to genes encoding IL-4 and IL-13 reduced the production of these cytokines in local (submandibular) lymph nodes. Suppression IL-4 and IL-13 production resulted in decrease in the levels of allergen-specific IgE- and IgG1-antibodies in the serum of mice and nasal hyperreactivity. A decrease in inflammation and upper respiratory tract remodeling was also observed after siRNA mediated suppression of IL-4 and IL-13 production.
Conclusion. Experimental therapy with siRNAs suppressing the production of pro-inflammatory cytokines IL-4 and IL-13 significantly alleviated the symptoms of allergic inflammation of the upper respiratory tract. Synthetic siRNAs have significant potential in the treatment of inflammatory diseases.
Changes of the expression of innate immunity receptors NOD-1, TLR8, ZBP-1, RIG-1 in myometrium during preeclampsia
Introduction. Preeclampsia (PE) is characterized by excessive and progressive activation of the immune system against the background of increased levels of pro-inflammatory cytokines and antiangiogenic factors in the placenta as well as in vascular endothelium in pregnant women. At the same time, researchers focus on the pathology of the placenta, while the state of the myometrium in this pathology remains unclear. Pattern recognition receptors (PRRs): Toll-like (TLRs), NOD-like (NLRs), RIG-like (RLRs) and DAI (DNA-binding protein 1, ZBP-1) play an important role in innate immunity and are expressed not only in immune, but also in non-immune cells. It is assumed that some of the clinical manifestations of PE are caused by common underlying inflammatory mechanisms regulated by PRRs.
Aim – to analyze the expression of TLR8, NOD-1, RIG-1 and ZBP-1 in myocytes, endothelium and mesenchymal fibroblast-like cells (FBC) in uterine wall during PE.
Material and methods. The study was performed on myometrial samples obtained during caesarean section (CS) from the anterior wall of the uterus of 22 women of reproductive age (18–43 years), at a period of 27–39 weeks of gestation, of which 12 patients were diagnosed with PE (6 patients had severe PE, 6 – moderate). The group of comparison consisted of 10 women with uncomplicated full-term pregnancy. An immunohistochemical (IHC) study was performed on serial sections of the uterus using polyclonal antibodies to NOD-1, TLR8, ZBP-1 (DLM/DAI), RIG-1.
Results. The study in the patients with uncomplicated pregnancy revealed a uniform expression of all the examined PRRs in smooth muscle cells, vascular endothelium and FBC. In PE we detected a pronounced irregularity in staining of myometrium. In moderate PE, an increase in the expression of ZBP-1 in the FBC and endothelium and NOD-1 in the endothelium was noted relative to the group of comparison. Severe PE, in turn, unlike moderate PE, was characterized by low expression of RIG-1 in the endothelium and FBC, decreased expression of NOD1, and increased expression of TLR8 in the vascular endothelium.
Conclusion. IHC features of innate immunity receptor expression in the myometrium are significantly altered in PE, providing new insights into the pathogenesis of PE.
Perspective of the use of M. leprae recombinant proteins in serodiagnostics of leprae
Introduction. Leprae is a chronic granulomatous disease caused by Mycobacterium leprae (M. leprae), affecting ectodermal derivatives (primarily peripheral nerves and skin), often leading to disability. The most important tool for laboratory control of leprae is immunodiagnostics. Using bioinformatics and comparative genomics, it was identified and cloned genes encoding proteins unique to the lepra pathogen and potentially the most immunogenic. It was proposed that recombinant candidate antigens unique to M. leprae, such as ML0050 and ML0576, could serve as appropriate targets for measuring specific cellular and humoral immunity to M. leprae.
Аim. In this work, we aimed to obtain recombinant M. leprae antigens ML0050, ML0576 and the ML0050–ML0576 fusion recombinant protein, conjugated with BSA, and evaluate their diagnostic potential for serodiagnosticis of leprae.
Material and methods. Molecular genetics and immunobiochemical methods were used. We used blood sera of leprosy and tuberculosis patients and also sera of healthy donors.
Results. The recombinant M. leprae antigens ML0050 and ML0576 and the recombinant fusion protein ML0050–ML0576 conjugated to bovine serum albumin were evaluated in the determination of human IgG, IgM and IgA antibodies against the M. leprae in an enzyme immunoassay and immunochromatographic assay with sera from 15 leprae patients, 25 tuberculosis patients and 30 healthy donors.
Conclusion. The results showed the possibility of using these recombinant proteins as antigens for serodiagnostics of leprae.
The role of costimulation blockade in transplantology: from experiment to clinic
The review presents an analysis of the current state of the problem of using costimulation inhibitors to prevent graft rejection. We have shown the role of costimulatory molecules in the development of transplant rejection, as well as the success of applying costimulation blockers in experimental models (mice, non-human primates) and in clinical trials.
The presented data indicate new approaches in the treatment of transplant rejection using costimulation blockers. These include inhibitors of interactions CD80/CD86 – CD28, CD40 – CD154, ICOS – ICOS-L, OX40 – OX40L, 4-1BB – 4-1BBL, etc.
It seems promising to use hematopoietic stem cell transplantation with costimulation blockers, the combined use of which can achieve donor chimerism with the formation of transplantation tolerance. This will improve the prognosis and quality of life of allograft recipients, and will completely cancel immunosuppressive therapy.
Age-associated diseases: the role of the inflammasome complex
Inflammatory aging (inflammaging) is one of the manifestations of immunosenescence and is considered as an important risk factor for morbidity and mortality among the elderly. An important role in the development of chronic sterile inflammation, which underlies age-related pathology, is attributed to chronic activation of pattern recognition receptors of the innate immunity system and their signaling pathways, primarily by endogenous ligands. When these ligands bind to the matching TLR, the NF-κB signaling pathway – which is considered as a key pathway for the development of inflammaging – is activated.
Stimulation of the NLR leads to the formation of inflammasomes, one of the functions of which is the processing of pro-inflammatory cytokines to a biologically active form, which is also an important factor in inflammaging, and underlies the pathogenesis of various chronic diseases.
This review addresses the role of the inflammasome complex in regards to immunopathogenesis of the following age-related diseases: Alzheimer’s disease, Parkinson’s disease, type II diabetes mellitus, atherosclerosis, joint diseases. New data frames the NLRP3 inflammasome as the most significant factor in the development of age-related diseases.
The review outlines the contribution of other inflammasome complexes to the development of age-related diseases. Further study of mechanisms of inflammasome-mediated inflammaging should allow for identification of potential targets for the treatment of age-related diseases.
Immune changes in synovial fluid in rheumatoid arthritis
Rheumatoid arthritis (RA) is a systemic autoimmune disease closely associated with synovial tissue proliferation, pannus formation in small joints such as the hands, wrists and feet, cartilage destruction and systemic complications such as pulmonary, cardiovascular, neurological and skeletal muscle lesions, glucocorticoid-induced osteoporosis and infections. The importance of confirming the diagnosis and determining local activity is given to the study of synovial fluid. A deep understanding of the pathological process in the joint in RA, characterized by changes in autoreactive CD4+ T cells, B cells, macrophages, inflammatory cytokines, chemokines and autoantibodies, has now been achieved, although much remains to be explored. This article provides an updated overview of the pathogenesis of RA, revealing even more therapeutic targets for the intra-articular pathological process.