Non-coding RNAs Analysis of Eosinophil Subtypes in Asthma

Allergic Asthma Clinical Trial

Official title:

Non-coding RNAs Analysis of Eosinophil Subtypes in Asthma

Clinical Trial Details — Status: Recruiting

Administrative data

• NCT number: NCT04542902
• Other study ID #: LAB-1/2020
• Status: Recruiting
• Phase: N/A
• Start date: October 1, 2020
• Est. completion date: October 1, 2023

Study information

• Verified date: August 2020
• Source: Lithuanian University of Health Sciences
• Contact: Kestutis Prof. Dr. Malakauskas
• Phone: +37037326737
• Email: kestutis.malakauskas[@]lsmuni.lt
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

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.

Description:

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.

Recruitment information / eligibility

• Status: Recruiting
• Enrollment: 80
• Est. completion date: October 1, 2023
• Est. primary completion date: October 1, 2023
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years to 70 Years

Eligibility

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 ß2 agonists 12 hours prior the study; 4) long acting ß2 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.

Related Conditions & MeSH terms

• Allergic Asthma
• Asthma
• Pulmonary Eosinophilia
• Severe Eosinophilic Asthma

Intervention

Biological:
• Dermatophagoides pteronyssinus allergen
Dermatophagoides pteronyssinus allergen is required to perform allergen bronchial challenge test.

Procedure:
• Blood sampling
An amount of a person's blood taken from their body for use in medical.
• Bronchial challenge with allergen
Bronchial challenge is performed with D. pteronyssinus allergen. Measurements of differences in eosinophils activity after allergen challenge.

Locations

Country	Name	City	State
Lithuania	Lithuanian University of Health Sciences, Pulmonology Department	Kaunas

Sponsors (1)

Lead Sponsor	Collaborator
Lithuanian University of Health Sciences

Country where clinical trial is conducted

Lithuania, 

References & Publications (10)

Barnig C, Alsaleh G, Jung N, Dembélé D, Paul N, Poirot A, Uring-Lambert B, Georgel P, de Blay F, Bahram S. Circulating Human Eosinophils Share a Similar Transcriptional Profile in Asthma and Other Hypereosinophilic Disorders. PLoS One. 2015 Nov 2;10(11):e0141740. doi: 10.1371/journal.pone.0141740. eCollection 2015. — View Citation

Mazzeo C, Cañas JA, Zafra MP, Rojas Marco A, Fernández-Nieto M, Sanz V, Mittelbrunn M, Izquierdo M, Baixaulli F, Sastre J, Del Pozo V. Exosome secretion by eosinophils: A possible role in asthma pathogenesis. J Allergy Clin Immunol. 2015 Jun;135(6):1603-13. doi: 10.1016/j.jaci.2014.11.026. Epub 2015 Jan 21. — View Citation

Mesnil C, Raulier S, Paulissen G, Xiao X, Birrell MA, Pirottin D, Janss T, Starkl P, Ramery E, Henket M, Schleich FN, Radermecker M, Thielemans K, Gillet L, Thiry M, Belvisi MG, Louis R, Desmet C, Marichal T, Bureau F. Lung-resident eosinophils represent a distinct regulatory eosinophil subset. J Clin Invest. 2016 Sep 1;126(9):3279-95. doi: 10.1172/JCI85664. Epub 2016 Aug 22. — View Citation

Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10513-8. doi: 10.1073/pnas.0804549105. Epub 2008 Jul 28. — View Citation

Perry MM, Tsitsiou E, Austin PJ, Lindsay MA, Gibeon DS, Adcock IM, Chung KF. Role of non-coding RNAs in maintaining primary airway smooth muscle cells. Respir Res. 2014 May 16;15:58. doi: 10.1186/1465-9921-15-58. — View Citation

Rodrigo-Muñoz JM, Cañas JA, Sastre B, Rego N, Greif G, Rial M, Mínguez P, Mahíllo-Fernández I, Fernández-Nieto M, Mora I, Barranco P, Quirce S, Sastre J, Del Pozo V. Asthma diagnosis using integrated analysis of eosinophil microRNAs. Allergy. 2019 Mar;74(3):507-517. doi: 10.1111/all.13570. Epub 2018 Oct 11. — View Citation

Rosenberg HF, Dyer KD, Foster PS. Eosinophils: changing perspectives in health and disease. Nat Rev Immunol. 2013 Jan;13(1):9-22. doi: 10.1038/nri3341. Epub 2012 Nov 16. Review. — View Citation

Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol. 2006;24:147-74. Review. — View Citation

Weller PF, Spencer LA. Functions of tissue-resident eosinophils. Nat Rev Immunol. 2017 Dec;17(12):746-760. doi: 10.1038/nri.2017.95. Epub 2017 Sep 11. Review. — View Citation

Zhu Y, Mao D, Gao W, Han G, Hu H. Analysis of lncRNA Expression in Patients With Eosinophilic and Neutrophilic Asthma Focusing on LNC_000127. Front Genet. 2019 Mar 19;10:141. doi: 10.3389/fgene.2019.00141. eCollection 2019. — View Citation

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Fold changes of ncRNA expression between eosinophils subtypes	Validated ncRNA expression of rEOS and iEOS in severe and non-severe eosinophilic asthma patients and healthy subjects.	From 6 to 12 months
Primary	ncRNA levels in rEOS- and iEOS-derived exosomes	Qualitative and quantitative selected ncRNA levels in rEOS- and iEOS-derived exosomes of all investigated groups.	From 12 to 18 months
Secondary	Fold changes of ncRNA profiles of distinct eosinophil subtypes	Non-validated whole ncRNA profiles of distinct eosinophil subtypes in severe and non-severe eosinophilic asthma patients and healthy subjects.	From 6 to 12 months
Secondary	The fold changes of rEOS and iEOS surface integrins expression	The gene expression of selected outer-membrane integrins in eosinophil subtypes.	From 6 to 12 months
Secondary	The fold changes of rEOS and iEOS eosinophilopoietins receptors expression	The gene expression of interleukin (IL)-5, IL-3, and granulocyte-macrophage colony-stimulating factor in eosinophil subtypes.	From 6 to 12 months
Secondary	The efficiency of iEOS and rEOS adhesion	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.	From 6 to 12 months
Secondary	iEOS and rEOS survival differences	Viable iEOS and rEOS number after an appropriate period of time in combined cell culture with ASM cells or pulmonary fibroblasts.	From 6 to 12 months
Secondary	Quantity of iEOS and rEOS synthesized reactive oxygen species	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.	From 6 to 12 months
Secondary	Apoptotic iEOS and rEOS number	The number of apoptotic iEOS and rEOS after an appropriate period of time in combined cell culture with ASM cells or pulmonary fibroblasts.	From 6 to 12 months
Secondary	Concentrations of iEOS and rEOS produced proteins in investigated subjects' body fluids.	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.	From 6 to 12 months
Secondary	iEOS and rEOS effect on airway smooth muscle cells or pulmonary fibroblasts proliferation	The quantity of ASM cells or pulmonary fibroblasts after several repeats of proliferation in the presence or absence of eosinophil subtypes.	From 6 to 12 months
Secondary	iEOS and rEOS effect on apoptotic ASM cells and pulmonary fibroblasts number	The number of apoptotic ASM cells and pulmonary fibroblasts after an appropriate period of time in combined cell culture with iEOS and rEOS.	From 6 to 12 months
Secondary	iEOS and rEOS effect on migration of ASM cells.	The migrated ASM cells number after an appropriate period of time in combined cell culture with iEOS and rEOS	From 6 to 12 months
Secondary	iEOS and rEOS effect on migration of pulmonary fibroblasts	The migrated pulmonary fibroblasts number after an appropriate period of time in combined cell culture with iEOS and rEOS	From 6 to 12 months
Secondary	iEOS and rEOS effect on ASM cells contractility	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.	From 6 to 12 months
Secondary	iEOS and rEOS effect on pulmonary fibroblasts contractility	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.	From 6 to 12 months
Secondary	iEOS and rEOS effect on fold changes of ASM cells and pulmonary fibroblasts proteins expression;	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	From 6 to 12 months
Secondary	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	From 6 to 12 months