IL-4 regulates neutrophilic pulmonary inflammation in a mouse model of bronchial asthma.

Neutrophilic pulmonary inflammation in asthmatics substantially exacerbates the severity of the disease leading to resistance to conventional corticosteroid therapy. Many studies established the involvement of Th1- and Th17-cells and cytokines produced by them (IFNg, IL-17A, IL-17F etc.) in neutrophilic pulmonary inflammation. Recent studies revealed that IL-4 - a Th2-cytokine regulates neutrophil effector functions and migration. It was showed that IL-4 substantially reduces neutrophilic inflammation of the skin in a mouse model of cutaneous bacterial infection and blood neutrophilia in a mouse model systemic bacterial infection. However, there are no data available regarding the influence of IL-4 on non-infectious pulmonary inflammation. In the current study we investigated the effects of IL-4 in a previously developed mouse model of neutrophilic bronchial asthma. We showed that systemic administration of IL-4 significantly restricts neutrophilic inflammation of the respiratory tract probably through the suppression of Th1-/Th17-immune responses and downregulation of CXCR2. Additionally, pulmonary neutrophilic inflammation could be alleviated by IL-4-dependant polarization of N2 neutrophils and M2 macrophages, expressing anti-inflammatory TGFß. Considering these, IL-4 might be used for reduction of exaggerated pulmonary neutrophilic inflammation and overcoming corticosteroid insensitivity of asthma patients.

[Pleiomorphism of the cytokine profile in nasal polyp tissue depending on the phenotype of chronic rhinosinusitis with nasal polyps]. / Pleiomorfizm tsitokinovogo profilya v tkani polipov v zavisimosti ot fenotipa polipoznogo rinosinusita.

Clinical phenotypes of chronic rhinosinusitis with nasal polyps (CRSwNP) are characterized with different inflammation patterns of mRNA expression of cytokines and depend on presence of allergic rhinitis (AR), atopic bronchial asthma (aBA) or nonatopic bronchial asthma (nBA). OBJECTIVE: To compare inflammation response in patients with different phenotypes of CRSwNP according to level secretion of the key cytokines in nasal polyp tissue. MATERIAL AND METHODS: 292 patients with CRSwNP were divided into four phenotypes: group 1 - CRSwNP without respiratory allergy (RA) and without BA; group 2a - CRSwNP+ AR with aBA; group 2b - CRSwNP+AR without aBA; group 3 - CRSwNP+nBA. Control group (n=36) included patients with hypertrophic rhinitis without atopy or BA. Using multiplex assay we defined the level of IL-1ß, IL-4, IL-5, IL-6, IL-13, IFN-γ, TGF-ß1, TGF-ß2, TGF-ß3 in nasal polyp tissue. RESULTS: The evaluation of cytokines levels in nasal polyps in different CRSwNP phenotypes showed a pleiotropy of different cytokine secretion depending on different comorbid pathology. In control group we estimated the lowest levels of all detected cytokines in comparison with other CRS groups. High levels of local proteins IL-5 and IL-13 and low levels of all isoform of TGF-ß characterized CRSwNP without RA and BA. The combination of CRSwNP with AR showed high levels of proinflammatory cytokines IL-6 and IL-1ß, and high levels of TGF-ß1 and TGF-ß2. The combination of CRSwNP with aBA estimated low levels of proinflammatory cytokines IL-1ß, IFN-γ; in case of CRS+nBA we determined the highest levels of TGF-ß1, TGF-ß2 and TGF-ß3 in nasal polyp tissue. CONCLUSIONS: Each CRSwNP phenotype is characterized by different mechanism of local inflammation. This underlies the necessity to diagnose BA and respiratory allergy among these patients. The evaluation of local cytokine profile in different CRSwNP phenotypes can help to determine the target anticytokine therapy for patients who has low efficacy of basic corticosteroid therapy.

Modern View of Neutrophilic Asthma Molecular Mechanisms and Therapy.

For a long time asthma was commonly considered as a homogeneous disease. However, recent studies provide increasing evidence of its heterogeneity and existence of different phenotypes of the disease. Currently, classification of asthma into several phenotypes is based on clinical and physiological features, anamnesis, and response to therapy. This review describes five most frequently identified asthma phenotypes. Neutrophilic asthma (NA) deserves special attention, since neutrophilic inflammation of the lungs is closely associated with severity of the disease and with the resistance to conventional corticosteroid therapy. This review focuses on molecular mechanisms of neutrophilic asthma pathogenesis and on the role of Th1- and Th17-cells in the development of this type of asthma. In addition, this review presents current knowledge of neutrophil biology. It has been established that human neutrophils are represented by at least three subpopulations with different biological functions. Therefore, total elimination of neutrophils from the lungs can result in negative consequences. Based on the new knowledge of NA pathogenesis and biology of neutrophils, the review summarizes current approaches for treatment of neutrophilic asthma and suggests new promising ways to treat this type of asthma that could be developed in future.

[Prospects For the Use of Peptides against Respiratory Syncytial Virus].

The human respiratory syncytial virus (RSV) is one of the most common viral pathogens that affects the lower respiratory tract and could be a reason of bronchiolitis and/or pneumonia. Currently, there are no available effective ways of treating the RSV infection. Attempts to develop preventive vaccine have been unsuccessful. The only therapeutic agent used for RSV treatment is virazole (ribavirin); however, it induces adverse effects. Medications based on neutralizing monoclonal antibodies, such as IGIV (Respigam), palivizumab (Synagis), and MEDI-524 (Numab), are under clinical trials; however, their use will be limited by their high cost. One of the promising approaches for antiviral therapy is the use of natural peptides (defensins and cathelicidins), or their synthetic analogs. The majority of currently described antiviral peptides are developed against the human immunodeficiency virus, the herpes simplex virus, and the influenza virus. At the same time, a body of experimental data evidencing anti-RSV activity of peptides has been accumulated. The main advantages of peptide drugs are their wide spectrum of antiviral activity and low toxicity. However, there are obstacles in implementing peptide-based drugs in clinical practice. Due to their low resistance to the action of serum proteases, most authors consider peptides promising only for local application. Given that RSV affects the epithelium of the respiratory tract, where the protease activity is lower than in the systemic circulation, it is possible to develop locally active peptide drugs, for example, as inhalation forms. Their stability could also be increased by the synthesis of dendrimer peptides and by the development of recombinant peptides as precursor proteins. Anti-RSV peptides can be divided into several groups: (1) attachment and/or fusion blockers; (2) peptides displaying direct virucidal activity, disrupting the viral envelope. Such peptides, which suppress early stages of the viral life cycle, are considered prophylactic agents. However, for several peptides, their immunoregulatory properties have been described, which opens the possibility for therapeutic use. This review summarizes the information on the antiviral properties of such peptides and mechanisms of their action and describes the prospects of the future development of antiviral peptides.

Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma.

BACKGROUND: Mouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype. METHODS: Female BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41-43 and 62-64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR. RESULTS: The OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group. CONCLUSION: We have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.

The Role of Interleukin-37 in the Pathogenesis of Allergic Diseases.

Cytokines of the interleukin-1 (IL-1) family play an important role in the realization of the protective functions of innate immunity and are the key mediators involved in the pathogenesis of a wide range of diseases, including various manifestations of allergy. The IL-1 family includes more than 11 members. However, the functions of many of them remain to be elucidated. Recently, new members of the IL-1 family have been discovered. In 2000, several independent research groups reported the discovery of a new interleukin of this family, which was named IL-37, or IL-1F7 (according to the new nomenclature). IL-37 was assigned to the IL-1 family based on its structural similarity with other members of this family. The study of its biological properties showed that its activity changes in inflammatory diseases, such as rheumatoid arthritis, psoriasis, as well as allergic diseases (allergic rhinitis, bronchial asthma, and atopic dermatitis). However, unlike most members of the IL-1 family, IL-37 acts as a negative regulator of inflammation. Activation of IL-37 suppresses inflammation, resulting in the suppression of inflammatory cytokines and chemokines, which in turn prevents infiltration of pro-inflammatory cells, mainly eosinophils and neutrophils. The exact molecular and cellular mechanisms of the anti-inflammatory effect of IL-37 in the development of allergic diseases (AD) have not been fully studied. This review summarizes and analyzes the accumulated experimental data on the role of IL-37 in the pathogenesis of AD, such as allergic rhinitis, bronchial asthma, and atopic dermatitis.

[Gene expression levels of cytokines in different phenotypes of CRSwNP]. / Uroven' ékspressii genov tsitokinov pri raznykh fenotipakh polipoznogo rinosinusita.

INTRODUCTION: Rhinosinusitis with nasal polyps (CRSwNP) is often followed by a range of comorbid states, influence of which on the course of the main pathology process remains insufficiently studied. PURPOSE: To study the gene expression level of cytokines potentially talking part in the development of inflammation in nasal polyps with different phenotypes of CRSwNP. MATERIAL AND METHODS: All the patients with CRSwNP were divided into 4 equal groups, 36 patients in each subgroup: group 1 - CRSwNP without comorbid pathology; group 2 - CRSwNP+atopy; group 3 - CRSwNP + non-allergic bronchial asthma (BA); control group 4 - 36 patients diagnosed with hypertrophic rhinitis without atopy and without bronchial asthma. Using the real-time polymerase chain reaction (Real-Time PCR) method, the study of expression level of mRNA genes coding proteins IL-1ß, IL-4, IL-5, IL-13, IL-17F, IL-25, IFN-y, TSLP in polyp tissue was conducted. RESULTS: The statistically proved difference of expression level of cytokines depending on the CRSwNP phenotype was educed. If CRSwNP and atopy were combined, the gene expression level of all studied cytokines was statistically higher than that of CRSwNP without comorbid pathology; and the expression level of IL-17F, IL-25 and TSLP was more intense that in the group of CRSwNP + BA. There was no difference between the patients with comorbid allergy and comorbid BA regarding the gene expression of IFN-y, IL-5 and IL-13 cytokines. Among different phenotypes of CRSwNP no difference in IL-1ß expression level was detected, which evidences of persisting inflammatory process, and the IL-4 gene expression level was lower than the detection level in all the groups. CONCLUSION: With different CRSwNP phenotypes different inflammatory patterns are detected, which indicates different character of the pathology process course among these groups of patients. Higher expression level of cytokine genes, which are a marker of epithelial damage of IL-25 and TSLP, is found only among the patients with CRSwNP and atopy. It suggests that forming of CRSwNP without comorbid pathology is connected with other pathologic mechanisms, not with the damage to epithelial barrier. If CRSwNP + BA and CRSwNP + atopy were combined, the expression level of IFN-y, IL-5, IL-13 and IL-17F genes was higher than the one in the group of patients with CRSwNP without comorbid pathology. In view of obtained data, all the patients with CRSwNP shall be screened for bronchial asthma and the allergy diagnostic shall be conducted.

Effect of lipopeptide structure on gene delivery system properties: Evaluation in 2D and 3D in vitro models.

Development of efficient biodegradable, environmentally responsive, biocompatible and non-toxic delivery system is needed for efficient gene delivery. As well known, properties of the vehicle are determined by the structure of carrier components. The aim of the current study was to estimate in vitro transfection efficacy of aliphatic di-, tri- and tetrapeptide-based cationic lipoplexes loaded with siRNA in function of a number of cationic groups using 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Physicochemical properties and cytotoxicity of the liposomes were found to be dependent upon a number of amino acid derivatives in an amphiphilic polar head. Uptake of liposomes loaded with nucleic acid (lipoplexes) and their localization in HEK293T cells was studied by confocal microscopy. The liposomes based on lipotripeptides had the highest transfection efficiency which was 20-fold higher than those fabricated from lipotetrapeptides.

The Role of Interleukin-33 in Pathogenesis of Bronchial Asthma. New Experimental Data.

Interleukin-33 (IL-33) belongs to the IL-1 cytokine family and plays an important role in modulating immune system by inducing Th2 immune response via the ST2 membrane receptor. Epithelial cells are the major producers of IL-33. However, IL-33 is also secreted by other cells, e.g., bone marrow cells, dendritic cells, macrophages, and mast cells. IL-33 targets a broad range of cell types bearing the ST2 surface receptor. Many ST2-positive cells, such as Th2 cells, mast cells, basophils, and eosinophils, are involved in the development of allergic bronchial asthma (BA). This suggests that IL-33 directly participates in BA pathogenesis. Currently, the role of IL-33 in pathogenesis of inflammatory disorders, including BA, has been extensively investigated using clinical samples collected from patients, as well as asthma animal models. In particular, numerous studies on blocking IL-33 and its receptor by monoclonal antibodies in asthma mouse model have been performed over the last several years; IL-33- and ST2-deficient transgenic mice have also been generated. In this review, we summarized and analyzed the data on the role of IL-33 in BA pathogenesis and the prospects for creating new treatments for BA.

The potential of anti-infectives and immunomodulators as therapies for asthma and asthma exacerbations.

Asthma is responsible for approximately 25,000 deaths annually in Europe despite available medicines that maintain asthma control and reduce asthma exacerbations. Better treatments are urgently needed for the control of chronic asthma and reduction in asthma exacerbations, the major cause of asthma mortality. Much research spanning >20 years shows a strong association between microorganisms including pathogens in asthma onset, severity and exacerbation, yet with the exception of antibiotics, few treatments are available that specifically target the offending pathogens. Recent insights into the microbiome suggest that modulating commensal organisms within the gut or lung may also be a possible way to treat/prevent asthma. The European Academy of Allergy & Clinical Immunology Task Force on Anti-infectives in Asthma was initiated to investigate the potential of anti-infectives and immunomodulators in asthma. This review provides a concise summary of the current literature and aimed to identify and address key questions that concern the use of anti-infectives and both microbe- and host-based immunomodulators and their feasibility for use in asthma.

The TLR4 Agonist Immunomax Affects the Phenotype of Mouse Lung Macrophages during Respiratory Syncytial Virus Infection.

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 IFNγ (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.

[Lactose-based glycoconjugates with variable spacers for design of liver-targeted liposomes]. / Glikokon"iugaty na osnove laktozy so speiserami razlichnoi dliny dlia sozdaniia transportnykh sistem k kletkam pecheni.

Asialoglycoprotein receptors are highly abundant on the hepatocyte surface and have specific binding sites for blood serum glycoproteins. Such discovery resulted in development of liver-targeted drug delivery systems because modification of the liposomal surface by carbohydrate derivatives results in an increase of endocytosis, which facilitates selective uptake of such systems by hepatocytes. In this study we have synthesized novel lactose derivatives containing a palmitic hydrophobic domain. They were used for modification of the liposome surface. Transfection activity of modified liposomes was analyzed on the HepG2 cell line (hepatocytes) and showed an increase in the transfection efficiency as compared to the non-modified liposomes. At the same time transfection activities of modified and non-modified liposomes were similar in the case of a non-hepatocyte cell line (293T). The novel lactose-based glycoconjugates may be a promising tool for developing efficient vectors for delivery of nucleic acids to hepatocytes.

[Physicochemical properties of lipopeptide-based liposomes and their complexes with siRNA]. / Fiziko-khimicheskie svoistva kompleksov molekul miRNK i liposom na osnove lipopeptidov.

siRNA/cationic liposome complexes are efficient systems for transmembrane delivery. The aim of this study was to prepare a novel complex consisted of lipotripeptide OrnOrnGlu(C16H33)2 and siRNA molecule and examined their physicochemical properties. Electron microscopy study has shown that the siRNA/liposome complex (m/m 1/10) tends to form sandwich-like structures that may protect nucleic acid from nuclease degradation. Photon correlation spectroscopy data indicate that the particle size increased after siRNA adding, but did not exceed 300 nm in diameter, while z-potential of lipoplexes decreased from 22 mV to 14 mV, compared to the empty liposomes thus indicating positive charge neutralization by negatively charged siRNA. These data allow to hypothesize that such size and total positive charge could provide efficient cellular uptake by endocytosis. That may have good prospects for gene silencing therapy.

[Anticytokine therapy of allergic asthma].

Bronchial asthma is a chronic inflammatory disease of the airways that is characterized by episodes of shortness of breath, expiratory dyspnea, cough, wheezing, and pulmonary emphysema. At the present time, asthma is a global public health problem and affects about 5% of the worldwide population. Although a wide range of anti-inflammatory drugs is available, uncontrolled or poorly controlled asthma is still a problem, requiring the development of novel therapeutic approaches. Intense studies of the molecular mechanisms of asthma in transgenic animals performed since the 1990s implicated cytokines, such as IL-4, IL-5, and IL-13, and their receptors in the initiation and maintenance of asthma. These findings led to anticytokine therapy as a novel approach for bronchial asthma treatment. To date, many preclinical and clinical studies have been performed in this field especially with drugs based on humanized monoclonal antibodies, soluble receptors, peptide inhibitors, etc. The review summarizes the data from preclinical and clinical studies of anti-cytokine therapeutics in humans.

Interleukins 1 and 6 as Main Mediators of Inflammation and Cancer.

The idea of a potential link between cancer and inflammation was first proposed by R. Virchow in the nineteenth century. However, clear evidence regarding a key role of inflammation in oncogenesis appeared only during the last decade. Now the tumor microenvironment is commonly considered as an obligatory and significant component of almost all types of cancer, and the cells infiltrating such microenvironment are a source of inflammatory cytokines. Such cytokines play a key role in regulating inflammation during both normal immune response and developing cancer. In this review, we explore the role of two inflammatory cytokines interleukin 1 and interleukin 6 in cancer development. These cytokines have pleiotropic effects on various cell types in the tumor microenvironment, particularly being able to regulate pro-oncogenic transcription factors NF-κB and STAT3. For this reason, such cytokines influence key parameters of oncogenesis, increasing cell resistance to apoptosis, proliferation of cancer cells, angiogenesis, invasion and malignancy as well as the ability of tumor cells to respond to anticancer therapy. Here we summarize novel experimental data regarding mechanisms underlying the interaction between chronic inflammation and malignant neoplasms.

Synthesis and evaluation of novel lipopeptide as a vehicle for efficient gene delivery and gene silencing.

Nucleic acid-based therapeutics have recently emerged as a new class of next generation agents for treatment and prevention of viral infection, cancer, and genetic disorders, but their wide use is limited by their relatively weak delivery into target cells. Usage of synthetic cationic amphiphiles with peptide hydrophilic domain as agents for non-viral gene delivery is an attractive approach. We developed the schemes for the synthesis of aliphatic peptides with different length of the hydrocarbon chains in hydrophobic domains and different amino acids in polar head. For the obtained derivatives we determined transfection efficiency, critical vesicle concentration, particle size, ζ-potential and aggregates stability. We have found that the transfection efficiency is increased if the ornithine is a part of polar head in an amphiphile. The most promising amphiphile for liposomal formation OrnOrnGlu(C16H33)2 was examined more carefully. It has been shown that the lipopeptide possesses low toxicity (in vitro and in vivo) and high transfection efficiency with pDNA and siRNA in different cell lines. In addition, the production of liposomes based on this lipopeptide is simple, quick and cheap. Thus OrnOrnGlu(C16H33)2 is a promising vehicle for gene delivery and gene silencing.