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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 < 0.05), and SEA eosinophil subtypes demonstrated the highest effect on sm-MHC, SM22, and COL1A1 gene expression. Moreover, AA and SEA patients’ blood eosinophil subtypes promoted migration of ASM cells and their ECM-related proliferation, compared with HS (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.

Massive hemoptysis is a serious medical condition or emergency which needs immediate treatment. It typically appears in the bronchial arteries and can be caused by a wide range of pulmonary diseases. This report is based on a very rare case of a patient bleeding from an anastomosis between the left phrenic artery and pulmonary arteries/veins. In this case, the chest computed tomography had detected changes which required doctors to perform a successful embolization.

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 < 0.05). Furthermore, rEOS-like cells had a more pronounced effect on reducing ASM cell apoptosis compared to that of iEOS-like cells (p < 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 < 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.

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.

Introduction: Eosinophilic inflammation is one of the main asthma features. Recently two eosinophil subtypes were identified: lung-resident eosinophils (rEOS) and inflammatory eosinophils (iEOS). However, their role in asthma pathogenesis is not entirely clear Aim: To investigate the effect of rEOS and iEOS on airway smooth muscle (ASM) cells proliferation and viability in asthma Methods: We involved 16 severe non-allergic eosinophilic asthma (SNEA) patients with a high dose of inhaled steroids, 8 steroid-free non-severe allergic asthma (AA) patients and 9 healthy subjects (HS). Total blood EOS were isolated by high-density centrifugation and magnetic separation, phenotyping - with antibodies against CD62L. An individual combined cell culture between EOS and immortalized ASM cells was prepared. ASM cells proliferation was measured by Alamar Blue assay; viability – by annexin V FITC and propidium iodide staining Results: Both EOS subtypes significantly increased proliferation of ASM cells, and the effect of rEOS was stronger compared with iEOS in all investigated groups: 42.6±12.5% vs. 21.6±7% in SNEA, 29.1±4.2% vs. 15.6±2.8% in AA, 11.8±3.0% vs. 3.9±1.8% in HS groups, p Conclusions: rEOS stronger enhanced proliferation of ASM cells and prolonged their viability, compared with the effect of iEOS in asthma

Introduction: Acute allergic asthma (AA) attack is associated with eosinophilic airway inflammation. We presumed that distinct eosinophil subtypes - lung-resident (rEOS) and inflammatory (iEOS) eosinophils, may differently respond during allergen provoked asthma episode Aim: To investigate the effect of allergen challenge to blood rEOS and iEOS ratio as well as their adhesion and viability in AA patients Methods: 8 steroid-free AA patients, sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) were investigated. Allergen challenge was performed for all study subjects. Blood EOS before and 24 h after allergen challenge were isolated by high-density centrifugation and magnetic separation, phenotyping - with antibodies against CD62L. EOS adhesion to airway smooth muscle (ASM) cells was measured by evaluating EOS peroxidase activity; EOS viability - by annexin V and PI staining Results: At baseline, the predominant eosinophil subtype in AA patients was iEOS with the rEOS:iEOS ratio (RIR) of 0.59 while in HS group RIR was 0.93. rEOS showed increased adhesion to ASM cells compared with iEOS by 14.7±2.9% in AA group and 17.2±2.0% in HS group (p Conclusion: Allergen challenge increased the RIR and prolonged iEOS viability without effect on adhesion of both EOS subtypes in AA patients

Introduction: Eosinophils (EOS) are the prominent inflammatory cells that participate in allergic asthma pathogenesis. Recently was found that EOS can be separated into two different subtypes: inflammatory EOS(iEOS) and resident EOS(rEOS). However, the effect of these EOS subtypes on ASM cell remodeling remains unclear. The aim was to compare iEOS and rEOS effect on ASM cells differentiation to contractile phenotype and migration in asthma Methods: We recruited 9 AA steroid-free patients and 6 healthy subjects (HS). The eosinophils were isolated from peripheral blood and iEOS were separated from rEOS using CD62L microbeads. The combined cultures with EOS and hTERT ASM cell lines were made. Gene expression of ASM cells contractile phenotype markers (smMHC, SM22, smMLCK) and for migration responsible integrin αV subunit in ASM cells were analyzed using qRT-PCR. ASM cells migration was measured using wound healing assay after 24,48,72h incubation with rEOS and iEOS Results: The genes expression of all ASM contractile phenotype markers were significantly higher after incubation with asthmatic rEOS compared to iEOS in both groups. Therefore, asthmatic rEOS had a significantly greater effect on these genes’ expression compared to healthy rEOS,p Conclusion: rEOS promoted the differentiation of ASM cells to contractile phenotype and migration more intensive than iEOS 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.

Enhanced contractility and migration of airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) are part of airway remodeling in asthma. Eosinophils are the central inflammatory cells that participate in airway inflammation. However, the role of asthmatic eosinophils in ASMC and PF contractility, migration, and differentiation to contractile phenotype has not yet been precisely described. A total of 38 individuals were included in this study: 13 steroid-free non-severe allergic asthma (AA) patients, 11 severe non-allergic eosinophilic asthma (SNEA) patients, and 14 healthy subjects (HS). For AA patients and HS groups, a bronchial allergen challenge with D. pteronyssinus was performed. Individual combined cell cultures were prepared from isolated peripheral blood eosinophils and immortalized ASMC or commercial PF cell lines separately. The migration of ASMC and PF was evaluated using wound healing assay and contractility using collagen gel assay. Gene expression of contractile apparatus proteins, COL1A1, COL5A1, and FN, in ASMC and PF was evaluated using qRT-PCR. We found that contractility and migration of ASMC and PF significantly increased after incubation with asthmatic eosinophils compared to HS eosinophils, p &lt, 0.05, and SNEA eosinophils demonstrated the highest effect on contractility of ASMC and migration of both cell lines, p &lt, 0.05. AA and SNEA eosinophils significantly increased gene expression of contractile apparatus proteins, COL1A1 and FN, in both cell lines, p &lt, 0.05. Furthermore, the allergen-activated AA eosinophils significantly increased the contractility of ASMC, and migration and gene expression in ASMC and PF, p &lt, 0.05. Thus, asthmatic eosinophils change ASMC and PF behavior by increasing their contractility and migration, contributing to airway remodeling.