Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma

BACKGROUND: Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. METHODS: A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. RESULTS: Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p < 0.05) in vitro. The expression of Wnt-5a, TGF-β1, collagen, and fibronectin genes in ASMC was significantly higher after 24 h of co-culture with eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p < 0.05). CONCLUSIONS: Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02648074

α4β1 and αMβ2 Integrin Expression and Pro-Proliferative Properties of Eosinophil Subtypes in Asthma

Eosinophilic inflammation is one of the main pathophysiological features in asthma. Two subtypes of eosinophils exist in the lung and systemic circulation: lung-resident eosinophils (rEOS) and inflammatory eosinophils (iEOS). We evaluated the expression of α4β1 and αMβ2 integrins of eosinophil subtypes and their influence on airway smooth muscle (ASM) cell proliferation and viability in asthma. We included 16 severe non-allergic eosinophilic asthma (SNEA) patients, 13 steroid-free, non-severe allergic asthma (AA) patients, and 12 healthy control subjects (HS). For AA patients, a bronchial allergen challenge with Dermatophagoides pteronyssinus was performed. The eosinophil subtypes were distinguished using magnetic bead-labeled antibodies against surface CD62L, and individual combined cell cultures were prepared with ASM cells. The integrins gene expression was analyzed by a quantitative real-time polymerase chain reaction. Proliferation was assessed by the Alamar blue assay, and viability by annexin V and propidium iodide staining. rEOS-like cells were characterized by the relatively higher gene expression of the β1 integrin subunit, whereas iEOS-like cells were characterized by the αM and β2 integrin subunits. The inclusion of either eosinophil subtypes in co-culture significantly increased the proliferation of ASM cells, and the effect of rEOS-like cells was stronger than iEOS-like cells (p &lt; 0.05). Furthermore, rEOS-like cells had a more pronounced effect on reducing ASM cell apoptosis compared to that of iEOS-like cells (p &lt; 0.05). Lastly, the bronchial allergen challenge significantly enhanced only the iEOS-like cells’ effect on ASM cell proliferation and viability in AA patients (p &lt; 0.05). These findings highlight the different expression of α4β1 and αMβ2 integrins on distinct eosinophil subtypes in asthma. Therefore, rEOS-like cells have a stronger effect in stimulating ASM cell proliferation and viability; however, contact with specific allergens mainly enhances pro-proliferative iEOS-like cell properties

Asthmatic Eosinophils Alter the Gene Expression of Extracellular Matrix Proteins in Airway Smooth Muscle Cells and Pulmonary Fibroblasts

The impaired production of extracellular matrix (ECM) proteins by airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) is a part of airway remodeling in asthma. This process might be influenced by eosinophils that migrate to the airway and abundantly secrete various cytokines, including TGF-β. We aimed to investigate the effect of asthmatic eosinophils on the gene expression of ECM proteins in ASMC and PF. A total of 34 study subjects were recruited: 14 with allergic asthma (AA), 9 with severe non-allergic eosinophilic asthma (SNEA), and 11 healthy subjects (HS). All AA patients underwent bronchial allergen challenge with D. pteronyssinus. The peripheral blood eosinophils were isolated using high-density centrifugation and magnetic separation. The individual cell cultures were made using hTERT ASMC and MRC-5 cell lines and the subjects’ eosinophils. The gene expression of ECM and the TGF-β signaling pathway was analyzed using qRT-PCR. We found that asthmatic eosinophils significantly promoted collagen I, fibronectin, versican, tenascin C, decorin, vitronectin, periostin, vimentin, MMP-9, ADAM33, TIMP-1, and TIMP-2 gene expression in ASMC and collagen I, collagen III, fibronectin, elastin, decorin, MMP-2, and TIMP-2 gene expression in PF compared with the HS eosinophil effect. The asthmatic eosinophils significantly increased the gene expression of several canonical and non-canonical TGF-β signaling pathway components in ASMC and PF compared with the HS eosinophil effect. The allergen-activated AA and SNEA eosinophils had a greater effect on these changes. In conclusion, asthmatic eosinophils, especially SNEA and allergen-activated eosinophils, imbalanced the gene expression of ECM proteins and their degradation-regulating proteins. These changes were associated with increased gene expression of TGF-β signaling pathway molecules in ASMC and PF

Blood Inflammatory-like and Lung Resident-like Eosinophils Affect Migration of Airway Smooth Muscle Cells and Their ECM-Related Proliferation in Asthma

Airway remodeling is a hallmark feature of asthma, and one of its key structural changes is increased airway smooth muscle (ASM) mass and disturbed extracellular matrix (ECM) homeostasis. Eosinophil functions in asthma are broadly defined; however, we lack knowledge about eosinophil subtypes&rsquo; interaction with lung structural cells and their effect on the airway&rsquo;s local microenvironment. Therefore, we investigated the effect of blood inflammatory-like eosinophils (iEOS-like) and lung resident-like eosinophils (rEOS-like) on ASM cells via impact on their migration and ECM-related proliferation in asthma. A total of 17 non-severe steroid-free allergic asthma (AA), 15 severe eosinophilic asthma (SEA) patients, and 12 healthy control subjects (HS) were involved in this study. Peripheral blood eosinophils were enriched using Ficoll gradient centrifugation and magnetic separation, subtyped by using magnetic separation against CD62L. ASM cell proliferation was assessed by AlamarBlue assay, migration by wound healing assay, and gene expression by qRT-PCR analysis. We found that blood iEOS-like and rEOS-like cells from AA and SEA patients&rsquo; upregulated genes expression of contractile apparatus proteins, COL1A1, FN, TGF-&beta;1 in ASM cells (p &lt; 0.05), and SEA eosinophil subtypes demonstrated the highest effect on sm-MHC, SM22, and COL1A1 gene expression. Moreover, AA and SEA patients&rsquo; blood eosinophil subtypes promoted migration of ASM cells and their ECM-related proliferation, compared with HS (p &lt; 0.05) with the higher effect of rEOS-like cells. In conclusion, blood eosinophil subtypes may contribute to airway remodeling by upregulating contractile apparatus and ECM component production in ASM cells, further promoting their migration and ECM-related proliferation, with a stronger effect of rEOS-like cells and in SEA

IL-5 and GM-CSF, but Not IL-3, Promote the Proliferative Properties of Inflammatory-like and Lung Resident-like Eosinophils in the Blood of Asthma Patients

Blood eosinophils can be described as inflammatory-like (iEOS-like) and lung-resident-like (rEOS-like) eosinophils. This study is based on the hypothesis that eosinophilopoetins such as interleukin (IL)-3 and IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) alter the proliferative properties of eosinophil subtypes and may be associated with the expression of their receptors on eosinophils. We investigated 8 individuals with severe nonallergic eosinophilic asthma (SNEA), 17 nonsevere allergic asthma (AA), and 11 healthy subjects (HS). For AA patients, a bronchial allergen challenge with Dermatophagoides pteronyssinus was performed. Eosinophils were isolated from peripheral blood using high-density centrifugation and magnetic separation methods. The subtyping of eosinophils was based on magnetic bead-conjugated antibodies against L-selectin. Preactivation by eosinophilopoetins was performed by incubating eosinophil subtypes with IL-3, IL-5, and GM-CSF, and individual combined cell cultures were prepared with airway smooth muscle (ASM) cells. ASM cell proliferation was assessed using an Alamar blue assay. The gene expression of eosinophilopoetin receptors was analyzed with a qPCR. IL-5 and GM-CSF significantly enhanced the proliferative properties of iEOS-like and rEOS-like cells on ASM cells in both SNEA and AA groups compared with eosinophils not activated by cytokines (p p < 0.05). In conclusion: IL-5 and GM-CSF promote the proliferative properties of iEOS-like and rEOS-like eosinophils; however, the effect of only IL-5 may be related to the expression of its receptors in asthma patients

Blood Inflammatory-like and Lung Resident-like Eosinophils Affect Migration of Airway Smooth Muscle Cells and Their ECM-Related Proliferation in Asthma

Airway remodeling is a hallmark feature of asthma, and one of its key structural changes is increased airway smooth muscle (ASM) mass and disturbed extracellular matrix (ECM) homeostasis. Eosinophil functions in asthma are broadly defined; however, we lack knowledge about eosinophil subtypes’ interaction with lung structural cells and their effect on the airway’s local microenvironment. Therefore, we investigated the effect of blood inflammatory-like eosinophils (iEOS-like) and lung resident-like eosinophils (rEOS-like) on ASM cells via impact on their migration and ECM-related proliferation in asthma. A total of 17 non-severe steroid-free allergic asthma (AA), 15 severe eosinophilic asthma (SEA) patients, and 12 healthy control subjects (HS) were involved in this study. Peripheral blood eosinophils were enriched using Ficoll gradient centrifugation and magnetic separation, subtyped by using magnetic separation against CD62L. ASM cell proliferation was assessed by AlamarBlue assay, migration by wound healing assay, and gene expression by qRT-PCR analysis. We found that blood iEOS-like and rEOS-like cells from AA and SEA patients’ upregulated genes expression of contractile apparatus proteins, COL1A1, FN, TGF-β1 in ASM cells (p p < 0.05) with the higher effect of rEOS-like cells. In conclusion, blood eosinophil subtypes may contribute to airway remodeling by upregulating contractile apparatus and ECM component production in ASM cells, further promoting their migration and ECM-related proliferation, with a stronger effect of rEOS-like cells and in SEA

Pirmosios Baltijos šalyse taikomos robotinės chirurgijos sistemos „Versius“ daugiadisciplinis diegimas

On June, 2023, Vilnius University Hospital Santaros Klinikos started robotic-assisted operations using VERSIUS robotic surgery system (CMR, Cambridge, UK) and performed 29 robotic surgeries within the first 3 weeks. This surgical system was purchased with funding from the European Regional Development Fund. Comprehensive three-month-long learning courses were organized for a total number of 32 persons, forming 8 independent teams, each consisting of 2 surgeons and 2 nurses. Learning courses consisted of online theoretical training modules, a one-week learning course including technical skills training with a simulator and dry runs as well as cadaveric surgical cases. Furthermore, an additional learning module included dry runs in the operating room prior to the first surgeries, following live and online proctored surgical cases. This enabled a rapid and smooth introduction of the robotic surgical system into clinical practice without putting patients at risk. Abdominal surgeons, urologists, and gynecologists have already performed surgeries using the VERSIUS surgical system and no major complications have been reported. Thoracic surgeons are underway to begin thoracic robotic-assisted surgeries, completing the multidisciplinary VERSIUS surgical system implementation process. Our goal is to share initial experience with robotic VERSIUS surgical system and evaluate its implementation in Vilnius University Hospital Santaros Klinikos.2023 m. birželio mėn. Vilniaus universiteto ligoninėje Santaros klinikose pradėtos atlikti robotinės operacijos, naudojant robotinės chirurgijos sistemą „Versius“ (CMR, Kembridžas, JK). Per pirmąsias tris savaites atliktos 29 robotinės operacijos. Sistema įsigyta panaudojus Europos regioninės plėtros fondo lėšas. Išsamius 3 mėn. teorinius kursus išklausė ir praktiniuose mokymuose dalyvavo 32 Santaros klinikų medicinos darbuotojai. Jie suformavo 8 nepriklausomas komandas, sudarytas iš 2 chirurgų ir 2 slaugytojų. Mokymosi kursai apėmė: nuotolinius teorinio mokymo modulius; vienos savaitės mokymosi kursą, kurio metu lavinti techniniai įgūdžiai naudojant virtualiosios realybės simuliatorius; techninių įgūdžių mokymus, naudojant pilvo ertmės ir krūtinės ląstos ertmės operacijų muliažus; praktinius seminarus, per kuriuos buvo atliekamos operacijos, naudojant kadaverinius preparatus. Artėjant pirmosioms operacijoms, visos 8 komandos išėjo antrąjį mokymosi kursą operacinėje, naudodamiesi chirurginių įgūdžių lavinimo ir operacijų simuliavimo muliažais. Mokymų kursų pabaigoje atliktos kruopščiai suplanuotos pirmosios robotinės operacijos, dalyvaujant patyrusiems robotinės chirurgijos specialistams – mentoriams. Kruopštus standartizuotas mokymosi procesas leido greitai ir sklandžiai įdiegti robotinę chirurgiją į klinikinę praktiką, nekeliant pavojaus pacientams. Naudodami chirurginę sistemą „Versius“, pilvo chirurgai, urologai ir ginekologai jau atliko reikšmingą kiekį operacijų, išvengdami didelių komplikacijų. Pastaruoju metu pirmąsias operacijas atliko ir krūtinės chirurgai. Taip Vilniaus universiteto ligoninėje Santaros klinikose baigiamas robotinės chirurgijos sistemos „Versius“ daugiadisciplinio diegimo procesas. Straipsnio tikslas – pasidalyti pirmąja patirtimi, naudojant „Versius“ robotinės chirurgijos sistemą, ir įvertinti šios sistemos diegimo Vilniaus universiteto ligoninėje Santaros klinikose procesą