Transcriptional response of macrophages to combined stimulation of a NOD- and a Toll-like receptor
Introduction. Activation of innate immune cells by agonists of two or more pattern-recognition receptors can result in mutual blunting or enhancement of activation signals. Based on expression of selected genes, it is assumed that combined stimulation of NOD- and Toll-like receptors results in synergistic enhancement of cellular response (the effect of agonist combination is greater than sum of effects of individual agonists). However, this assumption has not been tested using whole-transcriptome analysis.
Aim – to conduct whole transcriptome analysis of human macrophages activated by a combination of NOD1 and TLR4 receptor agonists in vitro.
Material and methods. Macrophages were obtained by culturing of healthy donor blood monocytes with granulocyte-macrophage colony-stimulating factors and stimulated by agonists of NOD1 and TLR4 separately or in combination for 1 and 4 h. Transcriptomes were evaluated using high-throughput RNA sequencing (RNA-seq) and real-time PCR (RT-PCR). To identify synergistically inducible genes, potentiation indices were calculated (response to agonist combination divided by sum of responses to separate agonists).
Results. Synergisticall inducible genes comprised no more than 10 % of all genes induced by NOD1 and/or TLR4 agonists separately or in combination. Typical features of synergistically inducible genes were low basal expression and high response to combined stimulation. Despite their relatively low number, this group of genes is of great functional importance, because it includes genes of key pro-inflammatory cytokines. Results of bioinformatic analysis point at the role of NF-κB and AP-1 transcription factor families in the regulation of expression of synergistically inducible genes.
Conclusion. Knowledge of characteristics and mechanisms of synergistic effects will allow to select appropriate targets to suppress excessive cytokine production during cytokine storm, as well as to choose appropriate agonist combinations for therapeutic application in clinical situations demanding enhancement of innate immune response.
Additive effect of synthetic small interfering RNAs targeted to respiratory syncytial virus
Introduction. The respiratory syncytial virus (RSV) is one of the most prevalent pathogens. It causes severe diseases of the upper and lower respiratory tract. Annually, millions of children worldwide at age under 5 years old are dying from RSV. Effective, safe, and affordable ways of RSV treatment have not yet been developed. Discovery of RNA-interference phenomenon – a mechanism of negative gene regulation by small interfering RNA (siRNA) molecules – opened new opportunities in the development of novel specific effective drugs, including antivirals.
The aim of this study was to develop siRNA molecules against vitally important RSV genes and study its antiviral activity in vitro.
Material and methods. The design of siRNAs was carried out in silico using bioinformatic software. Antiviral activity was studied in the in vitro experiments on HEp-2 cell culture. Cells were transfected with siRNAs followed by infection and measurement of viral load by quantitative PCR and titration on a monolayer of sensitive MA-104 cells.
Results. First of all, we showed that HEp-2 cells out of the three virus-susceptible cell lines were more suitable for testing siRNA antiviral activity, as they are easier to transfect. Out of four screened siRNAs designed against vitally important RSV genes (n and p genes) two variants (siN-745 – against the n gene and siP4 – against the p gene) exhibited the most pronounced antiviral effect, reducing the virus titer by more than 10 times. The simultaneous use of these two siRNA variants provides even more pronounced (additive) antiviral effect, reducing virus replication by almost 100 times. Perhaps, such effect could be due to the fact that the p and n genes play different roles in RSV life cycle. The p gene encodes the replication enzyme and the n gene encodes the nucleoprotein that protects the virus genome from degradation.
Conclusion. We have shown that the simultaneous use of two siRNAs targeted to conserved n and p genes of the virus makes it possible to achieve a more pronounced antiviral effect compared to application of individual siRNAs. The developed siRNA variants can be used in the composition of antiviral drugs against RSV.
Investigation of TNF receptor coexpression parameters as potential biomarkers of effective hospital therapy with rituximab in patients with rheumatoid arthritis
Introduction. One of the main mechanisms regulating the biological action of tumor necrosis factor (TNF) as a key proinflammatory cytokine initiating pathological changes in rheumatoid arthritis (RA) is a change in the coexpression and expression density of specific receptors (TNFR1 and TNFR2). The search and testing of biomarkers associated with the regulation of cytokine activity is necessary to predict response to therapy and develop personalized strategies for managing patients with RA.
The aim of the study was to identify the parameters of expression and coexpression of TNFR1/TNFR2 associated with an insufficient effect after a course of hospital therapy with rituximab in terms of maintaining a long-term low activity of RA.
Material and methods. The study of expression and coexpression parameters of type 1 and type 2 receptors to TNF on immunocompetent cells of patients with RA was carried out by multicolor flow cytometry. Logistic regression analysis was used to identify potential biomarkers in patients with relapse after effective inpatient therapy.
Results. Among the 144 studied parameters of TNFR1/TNFR2 expression, 13 indicators were identified for 4 subpopulations of immunocompetent cells, which can be used as potential predictive biomarkers of long-term maintenance of low disease activity after rituximab therapy. It has been shown that combinations of expression parameters make it possible to calculate the level of response to therapy with greater prognostic significance compared to standard anamnestic, clinical and laboratory parameters. The final model included the percentage of TNFR2+ cells among monocytes [OR 0.861 (0.727 : 0.952), p = 0.023] and the number of TNFR1 on T cells [OR 1.636 (95 % CI 1.174 : 3.262), p = 0.038] with a determination coefficient of 0.52.
Conclusion. Changes in the coexpression profiles of TNF receptors on immunocompetent cells from patients with rheumatoid arthritis are associated with the level of response to therapy and the stability of remission.
Transduction of human B-lymphocytes using adeno-associated viruses of various serotypes
Introduction. Adeno-associated virus (AAV) vectors are the leading platform for gene therapy in the treatment of various human diseases. Gene delivery to human B-lymphocytes using AAV vectors presents significant challenges, as they are resistant to AAV infection. Researches is being carried out to find effective methods to overcome this problem.
The aim of the study was to develop a method for delivering genes using AAV vectors to human B-lymphocytes in vitro.
Material and methods. B-lymphocytes were isolated from the peripheral blood of healthy donors by Ficoll-Verografin density gradient centrifugation with further enrichment by negative selection using magnetic beads. B-lymphocytes were stimulated in vitro with a lymphokine mixture containing IL-2, IL-4, BAFF, IL-21 and multimerized CD40L (mCD40L). After 24 hours, stimulated B-lymphocytes were infected with recombinant AAV carrying the green fluorescent protein reporter gene (GFP). 3 serotypes AAV2, AAV6 and AAV-DJ have been tested. 48 hours after infection, the proportion of transduced B-lymphocytes was determined. After 7 days, the number and phenotype of stimulated B-lymphocytes were determined by flow cytometry, and the secretion of Ig into the supernatant of cultured cells was assessed using the enzyme-linked immunosorbent assay. HEK293T cells were used as a transduction control.
Results. Serotypes AAV2, AAV6 and AAV-DJ efficiently and equally transduced HEK293T cells. Unstimulated B-lymphocytes were resistant to the action of AAV and did not show a noticeable fluorescence of the GFP reporter protein. B-lymphocytes stimulated with a mixture of lymphokines, which included IL-2, IL-4, BAFF, IL-21 and mCD40L, were efficiently transduced with AAV2, AAV6 and AAV-DJ. The highest expression of the transgene was provided by the AAV6 serotype. The transduced B-lymphocytes continued to actively proliferate, acquired the plasmablast phenotype and secreted Ig into the supernatant. In terms of their functional properties, the transduced B-lymphocytes did not differ from the control cells that were not exposed to infection. Thus, at moderate doses, AAV infection did not have a toxic effect on B-lymphocytes and did not affect the physiological parameters of B-cells. Efficient transduction was observed in both subpopulations of memory B-cells, both with switched (IgG+CD27+) and non-switched (IgM+CD27+) Ig synthesis.
Conclusion. Stimulation with a mixture of lymphokines is critical for efficient transgene transfer into human B-lymphocytes using rAAV vectors. In the transduction of B-lymphocytes the AAV6 serotype is the most effective.
Lectin pathway proteins deficiency of the complement system activation in the Arctic population
Introduction. The genetically determined defects in the lectin pathway of the complement system activation have various clinical consequences for a particular individual. The hypothesis of lectin pathway redundancy suggests that it is not required for the normal functioning of the immune system in modern humans other than early childhood.
The aim of the study was to analyze the prevalence of polymorphic variants FCN3 and MASP2 among newborns of the Siberian Arctic populations (Nenets and Dolgans-Nganasans) and Russians of Eastern Siberia along with genetic data on the frequency of genotypes and haplotypes for the genes of other lectin pathway proteins of the complement system (MBL and L-ficolin).
Material and methods. DNA samples from newborns represented by three populations, i.e. Nenets, Dolgans-Nganasans and Russians (Caucasoids) analyzed by RT-PCR.
Results. The prevalence of the del rs532781899 FCN3 variant, which associated with reduced production of H-ficolin, was found to be increased in Russians compared to the aboriginal population of the Nenets (p = 0.002). The G rs72550870 MASP2 allele, which associated with low activity of the serum protease MASP-2, is increased in Russians compared to the Nenets and Dolgans-Nganasans (p < 0.001 and p = 0.03, respectively). The deficient variant of the MBL2 haplotype is more common in Russians than in other populations (p < 0.001).
Conclusion. The results of our studies have confirmed a hypothesis that human evolution in populations with historically higher hygienic and medical protection at an early age was aimed at accumulating the genotypes associated with low lectin pathway activity of complement activation.
Pathogenetic significance of the IL-10 family in severe acne
Introduction. Currently, it has been established that acne is an inflammatory dermatosis with early subclinical formation of an inflammatory reaction. The family of cytokines associated with IL-10 includes several representatives: IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26 and type III interferons (IL-28A, IL-28B and IL-29). Currently, the mechanism of inflammation induction in acne is multicomponent, but not yet fully understood. At the same time, the role of the IL-10 family in the pathogenesis of acne has not been fully established.
Aim – to study the pathogenetic significance of the IL-10 family (IL-10, IL-19, IL-20, IL-22, IL-26) and type III interferons (IL-28A/IFN-λ2, IL-29/IFN-λ1) in severe acne.
Material and methods. To achieve this goal, a prospective open, non-randomized, single-center comparative study was conducted in 2020–2023. We observed 77 people aged 16 to 44 years in clinical conditions (median – 23.5 [10.7; 25.9] year). Studies of the levels of cytokines IL-10, IL-19, IL-20, IL-22, IL-26 and type III interferons (IL-28A/IFN-λ2, IL-29/IFN-λ1) were carried out in all 57 patients of the main group and 20 apparently healthy individuals of the comparison group in blood serum using multiplex enzyme immunoassay (Bio-Plex system using Luminex xMAP technology) using the Bio-Plex Pro™ Human Treg Cytokine Panel, 12-Plex (Bio-Rad Laboratories, USA). The results of the multiplex analysis were read on the Bio-Plex 200 System analyzer (Bio-Rad Laboratories, USA) using Luminex xPONENT software. Statistical processing of the results was carried out using software products Microsoft Excel 2016, GraphPad Prizm 5.0 and Statistica 10.0.
Results. The level of cytokines IL-20, IL-22, IL-26 in the blood serum of patients with severe acne was significantly (p < 0.05) increased relative the comparison group. At the same time, the level of cytokines IL-10 and IL-19, which have pronounced anti-inflammatory activity, significantly increased in the main group compared with the comparison group. Analysis of the level of type III interferons IL-28A/IFN-λ2 and IL-29/IFN-λ1 showed its significant increase (p < 0.05) in the sera of acne patients compared with the comparison group.
Conclusion. The data obtained by us on changes in the levels of cytokines of the IL-10 family (IL-10, IL-19, IL-20, IL-22, IL-26) and type III interferons (IL-28A/IFN-λ2, IL-29/IFN-λ1) indicate a violation of the interaction of innate and adaptive immunity systems in severe acne, which is important for understanding the mechanism of development of this disease.
Doubts and hopes of allergology
The main aspects of the development of an allergic reaction, evolutionary stages of allergy formation, effector cells and regulatory molecules involved in an allergic reaction are reviewed in the article. The biological role of allergy is discussed. The need for an in-depth study of the mechanisms of allergic processes is noted, which is the basis for the development of highly effective allergy control drugs and the creation of improved algorithms for allergen-specific immunotherapy.
Evaluation of the efficacy of different adjuvants in producing murine monoclonal antibodies to the receptor-binding domain of the S-protein of SARS-CoV-2
Introduction. The SARS-CoV-2-infection/COVID-19 pandemic began at the end of December 2019. In May 2023, the WHO leadership declared the end of the pandemic. The reason for this conclusion was the reduction of SARS-CoV-2 infection/COVID-19-caused mortality. However, the circulation of the virus continues, new mutations appear, new epidemiologically significant strains arise. Monoclonal antibodies (MAbs) are considered as potential drugs for the treatment or prevention of SARS-CoV-2 infection. To date, most potent neutralizing MAb aim to neutralize the receptor-binding domain (RBD) of S-protein of the SARS-CoV-2. There are many approaches to obtain MAb, but they are still a challenge. The correct choice of antigen and adjuvant plays a key role in the formation of an intense immune response. We performed a comparative evaluation of the efficacy of recombinant RBD and RBD fused to Fc fragment of immunoglobulin (RBD-Fc) emulsified with different adjuvants to achieve a high immune response in BALB/c mice.
The purpose of this study was to select the most effective antigen and adjuvant to increase the immune response in animals in order to obtain MAb.
Material and methods. Recombinant RBD and RBD fused to Fc-fragment of human immunoglobulin G1 were obtained for this work. In the next step, BALB/c mice were immunized according to nine schemes to obtain MAb specific to the RBD domain of the S-protein of SARS-CoV-2. On the third day after the last immunization blood was collected from each group of animals to determine the antibody specificity titer. Spleens were extracted from the group of hyperimmune mice to obtain a suspension of splenocytes for further fusion with Sp2/0-Ag14 partner cells. After fusion, cells were cultured and screened, cloned, and expanded upon monolayer formation.
Results. Comparative analysis of specific antibody titers to RBD in the blood of mice showed that the adjuvants aluminum hydroxide, complete Freund's adjuvant (CFA), aluminum hydroxide in combination with PAF and MagicTM Mouse have different abilities to induce an immune response when combined with recombinant RBD or RBD-Fc. The most effective immunogen was RBD-Fc emulsified with aluminum hydroxide and CFA, the antibody titer was 1 : 256,000. Seventeen hybrids were obtained by electrofusion, three of which were stable.
Conclusion. Adjuvant composition of aluminum hydroxide and IFA is the most effective for stimulation of immune response to obtain murine MAb to RBD domain of the S-protein of SARS-CoV-2. It is possible that this effect in enhancing immunogenicity was due to the mechanisms of action of the two adjuvants. IFA acted as an antigen depot due to the mineral oil in its composition, and aluminum hydroxide acted as antigen delivery. Since the molecular weight of the recombinant RBD protein was doubled, this allowed the aluminum hydroxide-based adjuvant to retain the antigen on its surface and provide it to the immune cells for a long time, stimulating the humoral immune response.
Immune checkpoint molecules as regulators of immune tolerance in pregnancy
The review presents current data on the immunoregulatory role of inhibitory receptors, which, together with ligands, are negative regulators of immune responses. The most studied molecules are CTLA-4 (CD152), PD-1 (CD279), Tim-3 (CD366), as well as Lag-3 (CD223), Tigit and VISTA. The interaction of checkpoint receptors with their ligands activates a number of mechanisms: it changes the ratio of Th1/Th2, Th17/Treg, and M1/M2 towards dominance of cells with regulatory activity; induces the generation of regulatory T-cells that suppress the proliferation of effector T-lymphocytes and stimulates the proliferation of decidual CD8+-T cells and the secretion of anti-inflammatory cytokines. Increased expression or co-expression of checkpoint molecules on T-lymphocytes leads to cell exhaustion, which significantly reduces the functional activity of T cells. According to modern research, inhibitory molecules play a significant role in maintaining a normal pregnancy. The review presents data on the expression of inhibitory molecules of decidual and peripheral T cells in all trimesters of pregnancy, which shows the essential importance of checkpoint receptors and ligands in the detection of tolerance to fetal alloantigens. At the same time, the onset of pregnancy is accompanied by an increase in the expression level of PD-1, CTLA-4, Tim-3 inhibitory molecules and their ligands on the decidual and peripheral cells, which affects the formation of physiological immunosuppression. The development of gestational complications, such as miscarriage and preeclampsia, is accompanied by impaired expression of checkpoint molecules both at the local and systemic levels.
Role of IL-25, IL-33 and TSLP in the development of corticosteroid resistance
Allergy are a common problem all over the world. These include allergic rhinitis, atopic dermatitis and, perhaps the most severe allergic disease, bronchial asthma. All of them have a major impact on quality of life and some are even life-threatening.
There are a number of protocols for the treatment of allergy, including treatment with glucocorticosteroids. However, it is worth noting that ~ 10 % of patients do not respond well to this type of therapy. In view of this, the search and study of new targets not only for the treatment of allergic diseases, but also for the prevention of the development of corticosteroid resistance in patients seems relevant. A number of cytokines described in the literature, including IL-25, IL-33 and TSLP are involved in the mechanism of allergic disease development, for example by contributing to Th2-cell activation and initiation of pro-inflammatory cascades, and some of them can influence the appearance of corticosteroid resistance. This review provides information on what we believe to be relevant therapeutic targets that would increase the efficacy of allergy therapy, including by reducing the risks of the development of said resistance.