Bronchom assuages airway hyperresponsiveness in house dust mite-induced mouse model of allergic asthma and moderates goblet cell metaplasia, sub-epithelial fibrosis along with changes in Th2 cytokines and chemokines.

Background: Asthma is a common obstructive airway disease with an inflammatory etiology. The main unmet need in the management of asthma is inadequate adherence to pharmacotherapy, leading to a poorly-controlled disease state, necessitating the development of novel therapies. Bronchom is a calcio-herbal formulation, which is purported to treat chronic asthma. The objective of the current study was to examine the in-vivo efficacy of Bronchom in mouse model of allergic asthma. Methods: Ultra high performance liquid chromatography was utilized to analyze the phytocompounds in Bronchom. Further, the in-vivo efficacy of Bronchom was evaluated in House dust mite (HDM)-induced allergic asthma in mice. Mice were challenged with aerosolized methacholine to assess airway hyperresponsiveness. Subsequently, inflammatory cell influx was evaluated in bronchoalveolar lavage fluid (BALF) followed by lung histology, wherein airway remodeling features were studied. Simultaneously, the levels of Th2 cytokines and chemokines in the BALF was also evaluated. Additionally, the mRNA expression of pro-inflammatory and Th2 cytokines was also assessed in the lung along with the oxidative stress markers. Results: Phytocompounds present in Bronchom included, gallic acid, protocatechuic acid, methyl gallate, rosmarinic acid, glycyrrhizin, eugenol, 6-gingerol and piperine. Bronchom effectively suppressed HDM-induced airway hyperresponsiveness along with the influx of leukocytes in the BALF. Additionally, Bronchom reduced the infiltration of inflammatory cells in the lung and it also ameliorated goblet cell metaplasia, sub-epithelial fibrosis and increase in α-smooth muscle actin. Bronchom decreased Th2 cytokines (IL-4 and IL-5) and chemokines (Eotaxin and IP-10) in the BALF. Likewise, it could also suppress the mRNA expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-33), and IL-13. Moreover, Bronchom restored the HDM-induced diminution of endogenous anti-oxidants (GSH and SOD) and the increase in pro-oxidants (GSSG and MDA). Furthermore, Bronchom could also decrease the nitrosative stress by lowering the observed increase in nitrite levels. Conclusion: Taken together, the results of the present study data convincingly demonstrate that Bronchom exhibits pharmacological effects in an animal model of allergic asthma. Bronchom mitigated airway hyperresponsiveness, inflammation and airway remodeling evoked by a clinically relevant allergen and accordingly it possesses therapeutic potential for the treatment of asthma.

[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

Stigmasterol alleviates airway inflammation in OVA-induced asthmatic mice via inhibiting the TGF-ß1/Smad2 and IL-17A signaling pathways.

Stigmasterol is a common dietary phytosterol with high nutritional value and physiological activity. In this study, we evaluated the effects of stigmasterol on inflammatory cytokines and the TGF-ß1/Smad2 and IL-17A signaling pathway in an ovalbumin (OVA)-induced asthma mouse model. Stigmasterol treatment improved airway remodeling. In addition, it significantly attenuated the symptoms of asthma attacks, reduced the number of macrophages, lymphocytes, neutrophils, and eosinophils in BALF and inflammatory cytokines, including IL-1ß, IL-5, IL-6, and IL-13. It further decreased the level of IL-17A in BALF, serum and spleen. Spleen single-cell suspension analysis via flow cytometry showed that IL-17A level was consistent with the results obtained in BALF, serum and spleen. Stigmasterol decreased the protein expression levels of TGF-ß, p-Smad2 and IL-17A in the spleen, by increasing the protein expression level of IL-10. After 24 h of co-culture of TGF-ß, IL-6 and stigmasterol, the level of IL-17 in CD4+ T cell supernatant was lower relative to levels in the group without stigmasterol. Meanwhile, stigmasterol treatment attenuated the expression level of TGF- ß, p-Smad2 and IL-17A proteins in CD4+ T cells and enhanced the expression levels of IL-10 protein. These data suggested that stigmasterol inhibited the TGF-ß1/Smad2 and IL-17A signaling pathway to achieve anti-asthmatic effects in the OVA-induced asthma mouse model. Collectively, the results of this study are that stigmasterol has achieved preliminary efficacy in the non-clinical laboratory, further studies are needed to consider the clinical application of stigmasterol.

Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma.

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFß1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Effects of azithromycin on bronchial remodeling in the natural model of severe neutrophilic asthma in horses.

Steroid resistance in asthma has been associated with neutrophilic inflammation and severe manifestations of the disease. Macrolide add-on therapy can improve the quality of life and the exacerbation rate in refractory cases, possibly with greater effectiveness in neutrophilic phenotypes. The mechanisms leading to these beneficial effects are incompletely understood and whether macrolides potentiate the modulation of bronchial remodeling induced by inhaled corticosteroids (ICS) is unknown. The objective of this study was to determine if adding azithromycin to ICS leads to further improvement of lung function, airway inflammation and bronchial remodeling in severe asthma. The combination of azithromycin (10 mg/kg q48h PO) and inhaled fluticasone (2500 µg q12h) was compared to the sole administration of fluticasone for five months in a randomized blind trial where the lung function, airway inflammation and bronchial remodeling (histomorphometry of central and peripheral airways and endobronchial ultrasound) of horses with severe neutrophilic asthma were assessed. Although the proportional reduction of airway neutrophilia was significantly larger in the group receiving azithromycin, the lung function and the peripheral and central airway smooth muscle mass decreased similarly in both groups. Despite a better control of airway neutrophilia, azithromycin did not potentiate the other clinical effects of fluticasone.

The impact of benzo[a]pyrene on murine allergic airway inflammation via epigenetic remodeling.

Simultaneous exposure to both BaP and house dust mites (HDM) has been shown to exacerbate pulmonary inflammation and hyperresponsiveness in a murine asthma model. The mechanistic insight into epigenetic inheritance for this effect, however, remains to be clarified. As such, in this study, we explore the molecular basis for the enhancement of asthma. Female BAL/C mice were intranasally administered HDM (25 µg in 25 µL saline) and/or BaP (10 µg/kg) every other day for 9 weeks. RNA sequencing and DNA methylation assessment were used to explore the underlying mechanism. Following simultaneous exposure to HDM and BaP, mice exhibited pulmonary inflammation and the transcript level of IL4i1b, muc4 and IL22ra2 that were associated with altered DNA methylation, suggesting that there may be an epigenetic basis for BaP-induced asthma exacerbation. Our data suggest that DNA methylation is a major epigenetic modification that accompanies airway remodeling associated with changes in the allergic mice.

Salidroside Attenuates Airway Inflammation and Remodeling via the miR-323-3p/SOCS5 Axis in Asthmatic Mice.

INTRODUCTION: Salidroside (Sal) a bioactive component extracted from Rhodiola rosea is remarkable for its anti-asthmatic effects. The study aimed to explore the molecular mechanism of Sal in airway inflammation and remodeling in asthmatic mice and provide a novel theoretical basis for asthma treatment. METHODS: An asthmatic mouse model was established via ovalbumin (OVA) treatment, followed by injection of Sal and transfection of miR-323-3p-mimic and sh- suppressor of cytokine signaling 5 (SOCS5). Expressions of miR-323-3p, SOCS5 mRNA, collagen (COL)-I, and COL-III were detected via reverse transcription quantitative polymerase chain reaction. SOCS5 protein level was detected via Western blot. Levels of IgE, IL-13, IL-4, and IL-5 were detected via enzyme-linked immunosorbent assay. Inflammatory cell infiltration was observed via hematoxylin-eosin staining. Collagen disposition was observed via Masson staining. Resistance index (RI) of airway hyperresponsiveness, and the number of total cells, inflammatory cells (eosinophil, macrophage, neutrophil, and lymphocyte) in bronchoalveolar lavage fluid (BALF) were observed. The binding relationship between miR-323-3p and SOCS5 was predicted through the RNA22 website and verified via dual-luciferase reporter assay. RESULTS: miR-323-3p was highly expressed in OVA-treated mice. Sal treatment reduced inflammatory cell infiltration, COL disposition, miR-323-3p expression, and IgE, IL-13, IL-4, IL-5, COL-I, and COL-III levels, RI value, and the number of total cells and inflammatory cells in BALF. miR-323-3p inhibited SOCS5 transcription. miR-323-3p overexpression or SOCS5 downregulation reversed the protecting role of Sal in asthmatic mice. CONCLUSION: Sal inhibited miR-323-3p expression to promote SOCS5 transcription, thereby attenuating airway inflammation and remodeling in asthmatic mice.

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Abstract Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases.

Sinomenine Relieves Airway Remodeling By Inhibiting Epithelial-Mesenchymal Transition Through Downregulating TGF-ß1 and Smad3 Expression In Vitro and In Vivo.

Airway remodeling is associated with dysregulation of epithelial-mesenchymal transition (EMT) in patients with asthma. Sinomenine (Sin) is an effective, biologically active alkaloid that has been reported to suppress airway remodeling in mice with asthma. However, the molecular mechanisms behind this effect remain unclear. We aimed to explore the potential relationship between Sin and EMT in respiratory epithelial cells in vitro and in vivo. First, 16HBE cells were exposed to 100 µg/mL LPS and treated with 200 µg/mL Sin. Cell proliferation, migration, and wound healing assays were performed to evaluate EMT, and EMT-related markers were detected using Western blotting. Mice with OVA-induced asthma were administered 35 mg/kg or 75 mg/kg Sin. Airway inflammation and remodeling detection experiments were performed, and EMT-related factors and proteins in the TGF-ß1 pathway were detected using IHC and Western blotting. We found that Sin suppressed cell migration but not proliferation in LPS-exposed 16HBE cells. Sin also inhibited MMP7, MMP9, and vimentin expression in 16HBE cells and respiratory epithelial cells from mice with asthma. Furthermore, it decreased OVA-specific IgE and IL-4 levels in serum, relieved airway remodeling, attenuated subepithelial collagen deposition, and downregulating TGF-ß1and Smad3 expression in mice with asthma. Our results suggest that Sin suppresses EMT by inhibiting IL-4 and downregulating TGF-ß1 and Smad3 expression.

Increased Monocyte-Derived CD11b+ Macrophage Subpopulations Following Cigarette Smoke Exposure Are Associated With Impaired Bleomycin-Induced Tissue Remodelling.

Rationale: The accumulation of macrophages in the airways and the pulmonary interstitium is a hallmark of cigarette smoke-associated inflammation. Notably, pulmonary macrophages are not a homogenous population but consist of several subpopulations. To date, the manner in which cigarette smoke exposure affects the relative composition and functional capacity of macrophage subpopulations has not been elucidated. Methods: Using a whole-body cigarette smoke exposure system, we investigated the impact of cigarette smoke on macrophage subpopulations in C57BL/6 mice using flow cytometry-based approaches. Moreover, we used bromodeoxyuridine labelling plus Il1a-/- and Il1r1-/- mice to assess the relative contribution of local proliferation and monocyte recruitment to macrophage accumulation. To assess the functional consequences of altered macrophage subpopulations, we used a model of concurrent bleomycin-induced lung injury and cigarette smoke exposure to examine tissue remodelling processes. Main Results: Cigarette smoke exposure altered the composition of pulmonary macrophages increasing CD11b+ subpopulations including monocyte-derived alveolar macrophages (Mo-AM) as well as interstitial macrophages (IM)1, -2 and -3. The increase in CD11b+ subpopulations was observed at multiple cigarette smoke exposure timepoints. Bromodeoxyuridine labelling and studies in Il1a-/- mice demonstrated that increased Mo-AM and IM3 turnover in the lungs of cigarette smoke-exposed mice was IL-1α dependent. Compositional changes in macrophage subpopulations were associated with impaired induction of fibrogenesis including decreased α-smooth muscle actin positive cells following intratracheal bleomycin treatment. Mechanistically, in vivo and ex vivo assays demonstrated predominant macrophage M1 polarisation and reduced matrix metallopeptidase 9 activity in cigarette smoke-exposed mice. Conclusion: Cigarette smoke exposure modified the composition of pulmonary macrophage by expanding CD11b+ subpopulations. These compositional changes were associated with attenuated fibrogenesis, as well as predominant M1 polarisation and decreased fibrotic activity. Overall, these data suggest that cigarette smoke exposure altered the composition of pulmonary macrophage subpopulations contributing to impaired tissue remodelling.

Lung developmental is altered after inhalation exposure to various concentrations of calcium arsenate.

Exposure to dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations. We have previously reported that inhalation of real-world mine tailing dusts during lung development can alter lung function and structure in adult male mice. These real-world dusts contain a mixture of metal(loid)s, including arsenic. To determine whether arsenic in inhaled dust plays a role in altering lung development, we exposed C57Bl/6 mice to a background dust (0 arsenic) or to the background dust containing either 3% or 10% by mass, calcium arsenate. Total level of exposure was kept at 100 µg/m3. Calcium arsenate was selected since arsenate is the predominant species found in mine tailings. We found that inhalation exposure during in utero and postnatal lung development led to significant increases in pulmonary baseline resistance, airway hyper-reactivity, and airway collagen and smooth muscle expression in male C57Bl/6 mice. Responses were dependent on the level of calcium arsenate in the simulated dust. These changes were not associated with increased expression of TGF-ß1, a marker of epithelial to mesenchymal transition. However, responses were correlated with decreases in the expression of club cell protein 16 (CC16). Dose-dependent decreases in CC16 expression and increases in collagen around airways was seen for animals exposed in utero only (GD), animals exposed postnatally only (PN) and animals continuously exposed throughout development (GDPN). These data suggest that arsenic inhalation during lung development can decrease CC16 expression leading to functional and structural alterations in the adult lung.

Nerve Growth Factor: A Potential Therapeutic Target for Lung Diseases.

The lungs play a very important role in the human respiratory system. However, many factors can destroy the structure of the lung, causing several lung diseases and, often, serious damage to people's health. Nerve growth factor (NGF) is a polypeptide which is widely expressed in lung tissues. Under different microenvironments, NGF participates in the occurrence and development of lung diseases by changing protein expression levels and mediating cell function. In this review, we summarize the functions of NGF as well as some potential underlying mechanisms in pulmonary fibrosis (PF), coronavirus disease 2019 (COVID-19), pulmonary hypertension (PH), asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Furthermore, we highlight that anti-NGF may be used in future therapeutic strategies.

Lipoxin A4 inhibits ovalbumin-induced airway inflammation and airway remodeling in a mouse model of asthma.

Asthma is a chronic respiratory disease, which is characterized by airway inflammation, remodeling and airway hyperresponsiveness. Airway remodeling is caused by long-term inflammation of the airways. Lipoxin A4 (LXA4) is a natural eicosanoid with powerful anti-inflammatory properties, and has been shown to serve a critical role in orchestrating pulmonary inflammation and airway hyper-responsiveness in asthmatic mice. However, its effect on airway remodeling is unknown. Female BALB/c mice were used to establish a mouse model of asthma which were sensitized and challenged by ovalbumin (OVA). LXA4 was intranasally administrated prior to the challenge. The results of our study indicated that LXA4 suppressed the OVA-induced inflammatory cell infiltration and T helper type 2 (Th2) cytokines secretion in the mouse model of asthma. Characteristics of airway remodeling, such as thickening of the bronchial wall and smooth muscle, overdeposition of collagen, and overexpression of α-smooth muscle actin (α-SMA) and collagen-I were reversed by LXA4. Furthermore, LXA4 suppressed the aberrant activation of the signal transducer and activator of transcription 3 (STAT3) pathway in the lung tissues of asthmatic mice. In conclusion, these findings demonstrated that LXA4 alleviated allergic airway inflammation and remodeling in asthmatic mice, which may be related to the inhibition of STAT3 pathway.

Anisakis pegreffii Extract Induces Airway Inflammation with Airway Remodeling in a Murine Model System.

Exposure of the respiratory system to the Anisakis pegreffii L3 crude extract (AE) induces airway inflammation; however, the mechanism underlying this inflammatory response remains unknown. AE contains allergens that promote allergic inflammation; exposure to AE may potentially lead to asthma. In this study, we aimed to establish a murine model to assess the effects of AE on characteristic features of chronic asthma, including airway hypersensitivity (AHR), airway inflammation, and airway remodeling. Mice were sensitized for five consecutive days each week for 4 weeks. AHR, lung inflammation, and airway remodeling were evaluated 24 h after the last exposure. Lung inflammation and airway remodeling were assessed from the bronchoalveolar lavage fluid (BALF). To confirm the immune response in the lungs, changes in gene expression in the lung tissue were assessed with reverse transcription-quantitative PCR. The levels of IgE, IgG1, and IgG2a in blood and cytokine levels in the BALF, splenocyte, and lung lymph node (LLN) culture supernatant were measured with ELISA. An increase in AHR was prominently observed in AE-exposed mice. Epithelial proliferation and infiltration of inflammatory cells were observed in the BALF and lung tissue sections. Collagen deposition was detected in lung tissues. AE exposure increased IL-4, IL-5, and IL-13 expression in the lung, as well as the levels of antibodies specific to AE. IL-4, IL-5, and IL-13 were upregulated only in LLN. These findings indicate that an increase in IL-4+ CD4+ T cells in the LLN and splenocyte resulted in increased Th2 response to AE exposure. Exposure of the respiratory system to AE resulted in an increased allergen-induced Th2 inflammatory response and AHR through accumulation of inflammatory and IL-4+ CD4+ T cells and collagen deposition. It was confirmed that A. pegreffii plays an essential role in causing asthma in mouse models and has the potential to cause similar effects in humans.

TIPE2 inhibits PDGF-BB-induced phenotype switching in airway smooth muscle cells through the PI3K/Akt signaling pathway.

BACKGROUND: Childhood asthma is a common respiratory disease characterized by airway inflammation. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) has been found to be involved in the progression of asthma. This study aimed to explore the role of TIPE2 in the regulation of airway smooth muscle cells (ASMCs), which are one of the main effector cells in the development of asthma. MATERIALS AND METHODS: ASMCs were transfected with pcDNA3.0-TIPE2 or si-TIPE2 for 48 h and then treated with platelet-derived growth factor (PDGF)-BB. Cell proliferation of ASMCs was measured using the MTT assay. Cell migration of ASMCs was determined by a transwell assay. The mRNA expression levels of calponin and smooth muscle protein 22α (SM22α) were measured using qRT-PCR. The levels of TIPE2, calponin, SM22α, PI3K, p-PI3K, Akt, and p-Akt were detected by Western blotting. RESULTS: Our results showed that PDGF-BB treatment significantly reduced TIPE2 expression at both the mRNA and protein levels in ASMCs. Overexpression of TIPE2 inhibited PDGF-BB-induced ASMC proliferation and migration. In addition, overexpression of TIPE2 increased the expression of calponin and SM22α in PDGF-BB-stimulated ASMCs. However, an opposite effect was observed with TIPE2 knockdown. Furthermore, TIPE2 overexpression blocked PDGF-BB-induced phosphorylation of PI3K and Akt, whereas the expression of p-PI3K and p-Akt were aggravated by TIPE2 knockdown. Additionally, the effects of TIPE2 overexpression and TIPE2 knockdown were altered by IGF-1 and LY294002 treatments, respectively. CONCLUSIONS: Our findings demonstrate that TIPE2 inhibits PDGF-BB-induced ASMC proliferation, migration, and phenotype switching via the PI3K/Akt signaling pathway. Thus, TIPE2 may be a potential therapeutic target for the treatment of asthma.

Family with sequence similarity 13 member A mediates TGF-ß1-induced EMT in small airway epithelium of patients with chronic obstructive pulmonary disease.

BACKGROUND: To explore the role of family with sequence similarity 13 member A (FAM13A) in TGF-ß1-induced EMT in the small airway epithelium of patients with chronic obstructive pulmonary disease (COPD). METHODS: Small airway wall thickness and protein levels of airway remodeling markers, EMT markers, TGF-ß1, and FAM13A were measured in lung tissue samples from COPD and non-COPD patients. The correlations of FAM13A expression with COPD severity and EMT marker expression were evaluated. Gain- and loss-of-function assays were performed to explore the functions of FAM13A in cell proliferation, motility, and TGF-ß1-induced EMT marker alterations in human bronchial epithelial cell line BEAS-2B. RESULTS: Independent of smoking status, lung tissue samples from COPD patients exhibited significantly increased small airway thickness and collagen fiber deposition, along with enhanced protein levels of remodeling markers (collagen I, fibronectin, and MMP-9), mesenchymal markers (α-SMA, vimentin, and N-cadherin), TGF-ß1, and FAM13A, compared with those from non-COPD patients. FAM13A expression negatively correlated with FEV1% and PO2 in COPD patients. In small airway epithelium, FAM13A expression negatively correlated with E-cadherin protein levels and positively correlated with vimentin protein levels. In BEAS-2B cells, TGF-ß1 dose-dependently upregulated FAM13A protein levels. FAM13A overexpression significantly promoted cell proliferation and motility in BEAS-2B cells, whereas FAM13A silencing showed contrasting results. Furthermore, FAM13A knockdown partially reversed TGF-ß1-induced EMT marker protein alterations in BEAS-2B cells. CONCLUSIONS: FAM13A upregulation is associated with TGF-ß1-induced EMT in the small airway epithelium of COPD patients independent of smoking status, serving as a potential therapeutic target for anti-EMT therapy in COPD.

Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine. In phase 2b and 3 studies, tezepelumab significantly reduced exacerbations versus placebo in patients with severe uncontrolled asthma, irrespective of baseline levels of type 2 inflammatory biomarkers. We investigated the mechanism of action of tezepelumab by assessing its effects on airway inflammatory cells, airway remodelling, and airway hyperresponsiveness. METHODS: CASCADE was an exploratory, double-blind, randomised, placebo-controlled, parallel-group, phase 2 study done in 27 medical centres in Canada, Denmark, Germany, the UK, and the USA. Adults aged 18-75 years with uncontrolled, moderate-to-severe asthma were randomly assigned (1:1) to receive tezepelumab 210 mg or placebo administered subcutaneously every 4 weeks for a planned 28 weeks, extended to up to 52 weeks if COVID-19-related disruption delayed participants' end-of-treatment assessments. Randomisation was balanced and stratified by blood eosinophil count. The primary endpoint was the change from baseline to the end of treatment in the number of airway submucosal inflammatory cells in bronchoscopic biopsy samples. Eosinophils, neutrophils, CD3+ T cells, CD4+ T cells, tryptase+ mast cells, and chymase+ mast cells were evaluated separately. This endpoint was also assessed in subgroups according to baseline type 2 inflammatory biomarker levels, including blood eosinophil count. Airway remodelling was assessed via the secondary endpoints of change from baseline in reticular basement membrane thickness and epithelial integrity (proportions of denuded, damaged, and intact epithelium). Exploratory outcomes included airway hyperresponsiveness to mannitol. All participants who completed at least 20 weeks of study treatment, had an end-of-treatment visit up to 8 weeks after the last dose of study drug, and had evaluable baseline and end-of-treatment bronchoscopies were included in the primary efficacy analysis. All participants who received at least one dose of study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, NCT03688074. FINDINGS: Between Nov 2, 2018, and Nov 16, 2020, 250 patients were enrolled, 116 of whom were randomly assigned (59 to tezepelumab, 57 to placebo). 48 in the tezepelumab group and 51 in the placebo group completed the study and were assessed for the primary endpoint. Treatment with tezepelumab resulted in a nominally significantly greater reduction from baseline to the end of treatment in airway submucosal eosinophils versus placebo (ratio of geometric least-squares means 0·15 [95% CI 0·05-0·41]; nominal p<0·0010), with the difference seen across all baseline biomarker subgroups. There were no significant differences between treatment groups in the other cell types evaluated (ratio of geometric least-squares means: neutrophils 1·36 [95% CI 0·94-1·97]; CD3+ T cells 1·12 [0·86-1·46]; CD4+ T cells 1·18 [0·90-1·55]; tryptase+ mast cells 0·83 [0·61-1·15]; chymase+ mast cells 1·19 [0·67-2·10]; all p>0·10). In assessment of secondary endpoints, there were no significant differences between treatment groups in reticular basement membrane thickness and epithelial integrity. In an exploratory analysis, the reduction in airway hyperresponsiveness to mannitol was significantly greater with tezepelumab versus placebo (least-squares mean change from baseline in interpolated or extrapolated provoking dose of mannitol required to induce ≥15% reduction in FEV1 from baseline: tezepelumab 197·4 mg [95% CI 107·9 to 286·9]; placebo 58·6 mg [-30·1 to 147·33]; difference 138·8 [14·2 to 263·3], nominal p=0·030). Adverse events were reported in 53 (90%) patients in the tezepelumab group and 51 (90%) patients in the placebo group, and there were no safety findings of concern. INTERPRETATION: The improvements in asthma clinical outcomes observed in previous studies with tezepelumab are probably driven, at least in part, by reductions in eosinophilic airway inflammation, as shown here by reduced airway eosinophil counts regardless of baseline blood eosinophil count. Tezepelumab also reduced airway hyperresponsiveness to mannitol, indicating that TSLP blockade might have additional benefits in asthma beyond reducing type 2 airway inflammation. FUNDING: AstraZeneca and Amgen.

Store-operated calcium entry-associated regulatory factor regulates airway inflammation and airway remodeling in asthma mice models.

Store-operated calcium entry (SOCE) is involved in the pathogenesis of airway inflammation and remodeling in asthma. Store-operated calcium entry-associated regulatory factor (SARAF) can downregulate SOCE. We sought to investigate the role of SARAF in the regulation of airway inflammation and remodeling in asthma mice models, as well as in the functional regulation of human airway smooth muscle cells (hASMCs). Balb/c mice were sensitized and challenged with ovalbumin to establish the asthma mice models. Mice were transfected with lentivirus, which expressed the SARAF gene + GFP (green fluorescence protein) or the negative control gene + GFP. Airway resistance was measured with the animal pulmonary function system. Airway inflammation and remodeling were evaluated via histological staining. In vitro cultured hASMCs were transfected with scrambled small interfering RNA (siRNA) or SARAF-specific siRNA, respectively. The proliferation, migration rate, hypertrophy, and SOCE activity of hASMCs were examined with Cell Counting Kit-8, wound healing test, bright field imaging, and Ca2+ fluorescence imaging, respectively. SARAF expression was measured by quantitative real-time PCR. Asthma mice models showed decreased SARAF mRNA expression in the lungs. SARAF overexpression attenuated airway inflammation, resistance, and also remodeling. Downregulation of SARAF expression with siRNA promoted the proliferation, migration, hypertrophy, and SOCE activity in hASMCs. SARAF plays a protective role against airway inflammation and remodeling in asthma mice models by blunting SOCE; SARAF may also be a functional regulating factor of hASMCs.

Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Curcumol Ameliorates Lung Inflammation and Airway Remodeling via Inhibiting the Abnormal Activation of the Wnt/ß-Catenin Pathway in Chronic Asthmatic Mice.

BACKGROUND: Curcumol exhibits anti-inflammatory effect, but its effect on chronic asthma lacked research. Therefore, this study explored the role of curcumol in asthma. METHODS: A chronic asthmatic mice model was established by ovalbumin induction. After treatment with curcumol, airway resistance in mice was detected by forced oscillation technique. The histopathological features of airway tissues, pulmonary inflammation, and inflammation cell recruitment in the bronchoalveolar lavage fluid (BALF) of mice were detected by hematoxylin-eosin staining. Collagen deposition in the airways of mice was examined by Masson staining. The secretion of ovalbumin-IgE, IL-4, IL-5, IL-13 in mouse serum and VEGFA secretion in BALF were analyzed by ELISA. Finally, the expressions of ß-catenin, Wnt5a, VEGFA, TGF-ß1, Fibronectin, and MMP-9 in mice lung tissues were determined by Western blot or immunohistochemical. RESULTS: Curcumol attenuated airway hyperresponsiveness, airway remodeling, and pulmonary inflammation in chronic asthmatic mice. Curcumol relieved collagen deposition in airway tissues, inflammation cell recruitment in BALF, and reduced the up-regulation of serum ovalbumin-IgE, IL-4, IL-5, and IL-13 and BALF VEGFA in chronic asthmatic mice. In addition, curcumol attenuated the up-regulated expressions of ß-catenin, Wnt5a, VEGFA, TGF-ß1, Fibronectin, and MMP-9 in the lung tissues of chronic asthmatic mice, but curcumol treatment did not show such effects on healthy mice. CONCLUSION: Our findings revealed that curcumol could ameliorate lung inflammation and airway remodeling by inhibiting the abnormal activation of the Wnt/ß-catenin pathway in chronic asthmatic mice, indicating that curcumol could be used as a novel anti-asthma drug for basic and clinical research.