Non-coding RNAs Analysis of Eosinophil Subtypes in Asthma

Chronic airway inflammation rich in eosinophils is an important feature seen in asthma. Airway and blood eosinophilia is associated with increased rates of asthma exacerbations and more intense treatment. Recently, the existence of two distinct eosinophils subtypes was revealed-lung-resident eosinophils (rEOS), which maturate independently to interleukin (IL) 5, with the primary function to maintain tissue homeostasis, and inflammatory eosinophils (iEOS), which mature in IL-5-dependent manner and are mainly involved in immune responses. Eosinophils' effect on the airway remodeling in asthma depends not only on the activity but also by their viable number in the lungs. Blood iEOS infiltrate the airways mainly after the environmental stimulus like allergen and leave the airways with bronchial secretions. However, rEOS reside lung tissue for their entire lifetime regulating local immunity. Blood rEOS and iEOS ratio alters in asthma, compared with healthy controls. It is known that the predominant eosinophils subtype in allergic asthma are iEOS, while rEOS are basic subtype in severe eosinophilic asthma patients, moreover, they are different in adhesive properties and survivability as well. Distinct biological properties allows to speculate about their different functions in asthma, however, there are still little information. Data about differently expressed microRNA (miRNA) profiles in eosinophils in asthma suggests, that eosinophils subtypes can be distinct in non-coding RNA (ncRNA) - microRNA (miRNA), piwi-interacting RNA (piRNA) and long non-coding RNA (IncRNA) profiles that could describe their role in asthma pathogenesis and act as biomarkers to discern asthma phenotypes.

Asthma is not cured, and only well-balanced treatment can control the course and severity of the disease. Most clinical symptoms rise from aberrant chronic airway inflammation mostly eosinophilic. Eosinophils are terminally differentiated granulocytes that actively contribute to innate and adaptive inflammatory cascades through the production and release of diverse chemokines, cytokines, lipid mediators and other growth factors. IL-5 plays a fundamental role in eosinophils maturation in the bone marrow, their recruitment, and activation at sites of inflammation. Historically eosinophils were described as a critical player in host defense, including parasites, viruses, fungi, or bacteria, giving them a destructive inflammatory cell label. However, it became clear that steady-state eosinophils can contribute to the immunoregulation and tissue homeostasis as well. Studies revealed that there are distinct eosinophils subtypes - immunoregulatory lung-resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), involved in immune responses. Distinct eosinophils subtypes with different functions determines the separate treatment. There are still only a few studies describing distinct eosinophils subtypes in the lungs or blood. It is the beginning of a new promising research area for better individualized eosinophilic asthma treatment, moreover, other eosinophilic diseases as well. Peripheral blood eosinophils studies are sufficiently relevant to the tissue eosinophils studies, as blood eosinophils are released into the bloodstream in a fully maturated form. Moreover, peripheral blood study could give additional information with possibilities to prevent eosinophils effects in the early stage, before migration to the airways. Furthermore, the existence of tissue-resident eosinophils in peripheral blood is confirmed and primary research for eosinophil subtypes surface markers was made according to the data of human blood eosinophils. Data about differently differently expressed microRNA (miRNA) profiles in eosinophils in asthma suggests, that eosinophils subtypes can be distinct in non-coding RNA (ncRNA) - microRNA (miRNA), piwi-interacting RNA (piRNA) and long non-coding RNA (IncRNA) profiles that could describe their role in asthma pathogenesis and act as biomarkers to discern asthma phenotypes. Researchers have plan to expand research by analyzing non-coding RNA (ncRNA) - miRNA, piRNA and lncRNA profiles of rEOS and iEOS as well as selected ncRNA signatures in blood plasma estimating their diagnostic value. Moreover, additional investigation of ncRNA in eosinophil-derived exosomes will provide important data about possible effect of eosinophils subtypes on airway remodeling via secreted ncRNA. ncRNAs are key regulators for gene transcription. However, there is evidence about their dysregulation in eosinophils during asthma. It will give important information about molecular signaling pathways that regulate the activity of distinct eosinophil subtypes during health and asthma, and provide the essential information about possible new therapeutic targets for their control. Additionally researchers will investigate the biological differences between rEOS and iEOS, including surface integrins and eosinophilopoietins receptors expression, adhesive properties, survivability, synthesized reactive oxygen species and apoptosis, as well as their effect on pulmonary structural cells physiological activity as proliferation, apoptosis, migration, contractility and proteins production, and will relate it with molecular signaling pathways, regulated by distinct expressed ncRNAs. ncRNAs can be stored in eosinophils exosomes and expressed to the surrounding environment. Information about ncRNAs in eosinophils-derived exosomes will demonstrate their function by affecting the other cells, especially after migration to airways. Moreover, ncRNAs are stable and resistant to blood RNases and differentially expressed in several pathologies. Researchers suppose that altered blood levels of ncRNAs could act as a possible new diagnostic biomarker in asthma.

Bronchial challenge is performed with D. pteronyssinus allergen. Measurements of differences in eosinophils activity after allergen challenge.

Validated ncRNA expression of rEOS and iEOS in severe and non-severe eosinophilic asthma patients and healthy subjects.

Qualitative and quantitative selected ncRNA levels in rEOS- and iEOS-derived exosomes of all investigated groups.

Non-validated whole ncRNA profiles of distinct eosinophil subtypes in severe and non-severe eosinophilic asthma patients and healthy subjects.

The gene expression of interleukin (IL)-5, IL-3, and granulocyte-macrophage colony-stimulating factor in eosinophil subtypes.

The differences in stable adhered iEOS and rEOS quantity in the combined cell culture with airway smooth muscle (ASM) cells or pulmonary fibroblasts compared between the investigated groups.

Viable iEOS and rEOS number after an appropriate period of time in combined cell culture with ASM cells or pulmonary fibroblasts.

Relative differences between iEOS and rEOS synthesized reactive oxygen species quantity after an appropriate period of incubation alone or with ASM cells or pulmonary fibroblasts.

The number of apoptotic iEOS and rEOS after an appropriate period of time in combined cell culture with ASM cells or pulmonary fibroblasts.

Selected iEOS and rEOS proteins concentrations, measured in investigated subjects body fluids, expressed as the amount of protein in the respective amount of fluid sample.

The quantity of ASM cells or pulmonary fibroblasts after several repeats of proliferation in the presence or absence of eosinophil subtypes.

The number of apoptotic ASM cells and pulmonary fibroblasts after an appropriate period of time in combined cell culture with iEOS and rEOS.

The migrated ASM cells number after an appropriate period of time in combined cell culture with iEOS and rEOS

The migrated pulmonary fibroblasts number after an appropriate period of time in combined cell culture with iEOS and rEOS

The relative efficiency of ASM cells' ability to contract collagen gel after an appropriate period of time in combined cell culture with iEOS and rEOS, expressed as reduced poured gel size in percentage, compared with control ASM cells without incubation with eosinophils.

The relative efficiency of pulmonary fibroblasts ability to contract collagen gel after an appropriate period of time in combined cell culture with iEOS and rEOS. expressed as reduced poured gel size in percentage, compared with control pulmonary fibroblasts, without incubation with eosinophils.

Altered selected ASM cells and pulmonary fibroblasts proteins expression, after incubation with iEOS and rEOS, expressed as fold changes in comparison with control cells, without incubation with eosinophils

iEOS and rEOS effect on fold changes of ASM cells and pulmonary fibroblasts proteins gene expression;

Altered selected ASM cells and pulmonary fibroblasts proteins gene expression, after incubation with iEOS and rEOS, expressed as fold changes in comparison with control cells, without incubation with eosinophils

Inclusion Criteria: Men and women between the ages of 18-70 years; Allergic asthma and sensitization to house dust mites (D. pteronyssinus) allergen, approved with: 1) medical history and symptoms more than one year; 2) skin prick test positive for D. pteronyssinus (positive wheals are those exceeding 3 mm in diameter greater than the negative control); 3) positive bronchial challenge with methacholine or documented reversible bronchial obstruction; Severe eosinophilic asthma; Premenopausal women if pregnancy test is negative; Healthy subjects without allergic and other chronic respiratory diseases (control group); Participants who gave his/her informed written consent. Exclusion Criteria: Asthma exacerbation 1 month prior to study; Clinically significant permanent allergy symptoms (ex. cat or dog dander induced allergy); Contraindications to perform an allergy skin test and/or bronchial provocation test: 1) active airway infection 1 month prior the study; 2) used medicaments: inhaled glucocorticoids intake 1 month prior the study, antihistamines intake 7 days prior the study; 3) short acting β2 agonists 12 hours prior the study; 4) long acting β2 agonists 2 days prior the study; 5) leukotriene receptor antagonists prior 14 days; Contraindications for epinephrine; Other significant mental and / or internal diseases and conditions, which could be as exclusion criteria due to the opinion of the researcher; Alcohol or narcotic abuse; Pregnancy; Breast-feeding.

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