Mast cell MrgprB2 in neuroimmune interaction in IgE-mediated airway inflammation and its modulation by ß-arrestin2.

Introduction: Allergic asthma has been linked to the activation of mast cells (MCs) by the neuropeptide substance P (SP), but the mechanism underlying this neuroimmune interaction is unknown. Substance P produced from cutaneous nociceptors activates MCs via Mas-related G-protein-coupled receptor B2 (MrgprB2) to enhance type 2 immune response in experimental atopic dermatitis in mice. We recently showed that the adapter protein ß-arrestin2 (ß-arr2) contributes to MrgprB2-mediated MC chemotaxis. The goals of this study were to determine if MrgprB2 facilitates neuroimmune interaction in IgE (FcεRI)-mediated allergic airway inflammation (AAI) and to assess if this response is modulated by ß-arr2. Methods: Wild-type (WT), MrgprB2-/- mice and mice with MC-specific deletion of ß-arr2 (Cpa3Cre+ /ß-arr2fl/fl ) were passively sensitized with anti-TNP-IgE and challenged with antigen. The generation of SP and MC recruitment in the lung were determined by immunofluorescence and toluidine blue staining, respectively. The transcripts for Tac1, MrgprB2, TNF-α, and Th2 cytokines in lung tissue were assessed by RT-PCR, and the release of selected cytokines in bronchoalveolar lavage (BAL) was determined by ELISA. Eosinophil and neutrophil recruitment in lung tissue and BAL were determined by immunofluorescence staining and flow cytometry, respectively. Goblet cell hyperplasia was determined by periodic acid-Schiff staining. Results: Following IgE sensitization and antigen challenge in WT mice, SP generation, and MC recruitment, transcripts for Tac1, MrgprB2, TNF-α, and Th2 cytokine were upregulated when compared to the control challenge. TNF-α, Th2 cytokine production, eosinophil/neutrophil recruitment, and goblet cell hyperplasia were also increased. These responses were significantly reduced in MrgprB2-/- and Cpa3Cre+ /ß-arr2fl/fl mice. Discussion: The data presented herein suggest that SP-mediated MrgprB2 activation contributes to AAI and goblet cell hyperplasia in mice. Furthermore, these responses are modulated by ß-arr2, which promotes MC recruitment to facilitate their activation through FcεRI.

Induction of chronic asthma up regulated the transcription of senile factors in male rats.

BACKGROUND: The main characteristic of asthma is chronic inflammation. We examined cellular senescence by histology and molecular assay in the lungs of a rat model of asthma. This model comprises sensitization by several intraperitoneal injections of ovalbumin with aluminium hydroxide, followed by aerosol challenges every other day. RESULTS: Data showed that asthma induction caused histological changes including, hyperemia, interstitial pneumonia, fibrinogen clots, and accumulation of inflammatory cells in the pleura. There is an elevation of IL-1ß and NF-kB proteins in the asthmatic group (P < 0.001) compared to the control group. The expression of ß-galactosidase increased (P < 0.01), while the expression of Klotho and Sox2 genes was decreased in the lung tissue of the asthmatic group (P < 0.01). CONCLUSION: Taken together, these findings suggest that asthmatic conditions accelerated the cellular senescence in the lung tissue.

Biologics in Asthma: Role of Biomarkers.

Our modern understanding of asthma mainly concerns identification of inflammatory endotype to guide management. The distinction mostly concerns identification of type-2 inflammation, for which different biomarkers have been well characterized. Blood eosinophils corroborate activity in the interleukin (IL)-5 axis while fraction of exhaled nitric oxide is indicative of the IL-4/IL-13 axis, giving us an indication of activity in these distinct but complementary pathways. These biomarkers predict disease activity, with increased risk of exacerbations when elevated, and a further, multiplicative increase when both are elevated. Serum immunoglobulin E is also implicated in this pathway, and can represent allergen-related stimulation.

Multifaceted roles of mitochondria in asthma.

Mitochondria are essential organelles within cells, playing various roles in numerous cellular processes, including differentiation, growth, apoptosis, energy conversion, metabolism, and cellular immunity. The phenotypic variation of mitochondria is specific to different tissues and cell types, resulting in significant differences in their function, morphology, and molecular characteristics. Asthma is a chronic, complex, and heterogeneous airway disease influenced by external factors such as environmental pollutants and allergen exposure, as well as internal factors at the tissue, cellular, and genetic levels, including lung and airway structural cells, immune cells, granulocytes, and mast cells. Therefore, a comprehensive understanding of the specific responses of mitochondria to various external environmental stimuli and internal changes are crucial for elucidating the pathogenesis of asthma. Previous research on mitochondrial-targeted therapy for asthma has primarily focused on antioxidants. Consequently, it is necessary to summarize the multifaceted roles of mitochondria in the pathogenesis of asthma to discover additional strategies targeting mitochondria in this context. In this review, our goal is to describe the changes in mitochondrial function in response to various exposure factors across different cell types and other relevant factors in the context of asthma, utilizing a new mitochondrial terminology framework that encompasses cell-dependent mitochondrial characteristics, molecular features, mitochondrial activity, function, and behavior.

Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro.

BACKGROUND: Chronic airway disease (CAD) is characterized by chronic airway inflammation and colonization of the lungs by pro-inflammatory pathogens. However, while various other bacterial species are present in the lower airways, it is not fully understood how they influence inflammation. We aimed to identify novel anti-inflammatory species present in lower airway samples of patients with CAD. METHODS: Paired sputum microbiome and inflammatory marker data of adults with CAD across three separate cohorts (Australian asthma and bronchiectasis, Scottish bronchiectasis) was analyzed using Linear discriminant analysis Effect Size (LEfSE) and Spearman correlation analysis to identify species associated with a low inflammatory profile in patients. RESULTS: We identified the genus Aggregatibacter as more abundant in patients with lower levels of airway inflammatory markers in two CAD cohorts (Australian asthma and bronchiectasis). In addition, the relative abundance of Aggregatibacter was inversely correlated with sputum IL-8 (Australian bronchiectasis) and IL-1ß levels (Australian asthma and bronchiectasis). Subsequent in vitro testing, using a physiologically relevant three-dimensional lung epithelial cell model, revealed that Aggregatibacter spp. (i.e. A. actinomycetemcomitans, A. aphrophilus) and their cell-free supernatant exerted anti-inflammatory activity without influencing host cell viability. CONCLUSIONS: These findings suggest that Aggregatibacter spp. might act to reduce airway inflammation in CAD patients.

Oleic acid attenuates asthma pathogenesis via Th1/Th2 immune cell modulation, TLR3/4-NF-κB-related inflammation suppression, and intrinsic apoptotic pathway induction.

WHO reported that asthma was responsible for 455,000 deaths in 2019 and asthma patients was evaluated 262 million in May 2023. The incidence is expected to increase as the average life expectancy increases, highlighting asthma as a significant health challenge in an aging society. The etiology of asthma is linked to an imbalance of Th1 and Th2 cells, respiratory inflammation, and pulmonary cell proliferation. The purpose of this study is to investigate the anti-asthmatic effect and potential mechanism of oleic acid. The anti-inflammatory effect of oleic acid was evaluated in an LPS-induced RAW 264.7 cell model, and immune modulation and the anti-apoptotic effect were measured in an ovalbumin-induced BALB/c mouse model. A variety of analytical procedures, such as MTT, qPCR, ELISA, Western blotting, immunofluorescence, gene transfection, immunohistochemistry, and several staining methods (Diff Quik, H&E, PAS), were used to evaluate the effectiveness and mechanisms of these methods. The results from in vitro experiments showed that oleic acid could reduce the levels of inflammatory cytokines (TNF-α, IL-6, and IL-1ß), and molecular docking studies suggested that oleic acid could interact with TLR3 and TLR4 proteins to form ligand-protein complexes, showing good binding affinity. Additionally, oleic acid attenuated the expression of MAPK pathway components (JNK, p38 MAPK) and NF-κB pathway constituents (IκB, NF-κB, COX-2, PGE2). In vivo results indicated that oleic acid reduced the levels of inflammatory cells (WBCs and eosinophils) and IgE activity, reduced the expression of the Th2 cell transcription factor GATA-3, and decreased the levels of Th2/Th17-related cytokines (IL-4, TNF-α, and IL-6). Oleic acid also alleviated OVA-induced pathological changes in the lung, such as epithelial cell proliferation, inflammatory cell infiltration, and mucus hypersecretion. OVA restored apoptosis in lung epithelial cells by modulating the expression of Bcl-2 and Bax. In summary, oleic acid has potential as a novel candidate for asthma treatment through its ability to regulate immune cells, exert anti-inflammatory effects, and promote apoptosis, thereby ameliorating asthma manifestations.

Drosophila melanogaster as an Alternative Model to Higher Organisms for In Vivo Lung Research.

COPD and asthma are lung diseases that cause considerable burden to more than 800 million people worldwide. As both lung diseases are so far incurable, it is mandatory to understand the mechanisms underlying disease development and progression for developing novel therapeutic approaches. Exposures to environmental cues such as cigarette smoke in earliest life are known to increase disease risks in the individual's own future. To explore the pathomechanisms leading to later airway disease, mammalian models are instrumental. However, such in vivo experiments are time-consuming and burdensome for the animals, which applies in particular to transgenerational studies. Along this line, the fruit fly Drosophila melanogaster comes with several advantages for research in this field. The short lifespan facilitates transgenerational studies. A high number of evolutionary conserved signaling pathways, together with a large toolbox for tissue-specific gene modification, has the potential to identify novel target genes involved in disease development. A well-defined airway microbiome could help to untangle interactions between disease development and microbiome composition. In the following article, Drosophila melanogaster is therefore presented and discussed as an alternative in vivo model to investigate airway diseases that can complement and/or replace models in higher organisms.

Nobiletin, as a Novel PDE4B Inhibitor, Alleviates Asthma Symptoms by Activating the cAMP-PKA-CREB Signaling Pathway.

Asthma is a chronic airway inflammation that is considered a serious public health concern worldwide. Nobiletin (5,6,7,8,3',4'-hexamethyl flavonoid), an important compound isolated from several traditional Chinese medicines, especially Citri Reticulatae Pericarpium, is widely used for a number of indications, including cancer, allergic diseases, and chronic inflammation. However, the mechanism by which nobiletin exerts its anti-asthmatic effect remains unclear. In this research, we comprehensively demonstrated the anti-asthmatic effects of nobiletin in an animal model of asthma. It was found that nobiletin significantly reduced the levels of inflammatory cells and cytokines in mice and alleviated airway hyperresponsiveness. To explore the target of nobiletin, we identified PDE4B as the target of nobiletin through pharmacophore modeling, molecular docking, molecular dynamics simulation, SPR, and enzyme activity assays. Subsequently, it was found that nobiletin could activate the cAMP-PKA-CREB signaling pathway downstream of PDE4B in mouse lung tissues. Additionally, we studied the anti-inflammatory and anti-airway remodeling effects of nobiletin in LPS-induced RAW264.7 cells and TGF-ß1-induced ASM cells, confirming the activation of the cAMP-PKA-CREB signaling pathway by nobiletin. Further validation in PDE4B-deficient RAW264.7 cells confirmed that the increase in cAMP levels induced by nobiletin depended on the inhibition of PDE4B. In conclusion, nobiletin exerts anti-asthmatic activity by targeting PDE4B and activating the cAMP-PKA-CREB signaling pathway.

Acupoint Autohemotherapy Alleviates Airway Inflammation in Asthmatic Rats via Upregulating Expression of Hemeoxygenase-1.

Importance: Acupoint autohemotherapy (AA), a therapeutic technique involving the subcutaneous injection of autologous blood into acupoints, has been empirically validated as safe and effective for treating asthma by alleviating symptoms and decreasing acute attacks, though its mechanism is not well understood. Objective: The role of heme oxygenase-1 (HO-1) in AA-induced suppression of asthmatic airway inflammation is examined. Methods: Twenty rats were assigned randomly to four groups, namely the Control, OVA, OVA + AA, and (OVA + Snpp) + AA. Rats in the OVA + AA and (OVA + Snpp) + AA received autologous blood injections into acupoints (BL13 and BL23) following OVA challenge. Rats in the (OVA + Snpp) + AA were concurrently subjected to intraperitoneal injections of Snpp, a inhibitor of HO-1. Airway inflammation was evaluated through HE staining, while the concentrations of cytokines in BALF were quantified using ELISA. The mRNA and protein levels of RORγt (Th17-specific transcription factor), Foxp3 (Treg-specific transcription factor), and HO-1 in lung tissue were assessed through qRT-PCR and WB. Results: HE staining indicated that airway inflammation was alleviated in the OVA + AA. The OVA + AA displayed significantly lower counts of total cells and eosinophils in the BALF compared to both the OVA and (OVA + Snpp) + AA. The ELISA demonstrated a significant decrease in levels of pro-inflamatory cytokines (IL-4, IL-17A), and an increase in levels of anti-inflamatory cytokines (IFN-γ, IL-10), in the OVA + AA when compared to both OVA and (OVA + Snpp) + AA. The qRT-PCR and WB analyses revealed an upregulation of HO-1 and Foxp3 expression, and a downregulation of RORγt expression, in the OVA + AA when compared to OVA and (OVA + Snpp) + AA. Conclusions and Relevance: The involvement of HO-1 in the underlying mechanism responsible for the anti-inflammatory effects of AA is evident.

Inhibition of long non-coding RNA NEAT1 suppressed the epithelial mesenchymal transition through the miR-204-5p/Six1 axis in asthma.

BACKGROUND: Asthma, a prevalent chronic respiratory condition, is characterized by airway remodeling. Long non-coding RNA (lncRNA) NEAT1 has been demonstrated to participate in airway fibrosis. Furthermore, the miR-204-5p/Six1 axis significantly influences epithelial mesenchymal transition (EMT). However, the function of NEAT1/miR-204-5p/Six1 in asthmatic EMT remains unclear. PURPOSE: This study intends to elucidate the function of NEAT1/miR-204-5p/Six1 axis in asthmatic EMT. METHODS: TGF-ß1 was used to induce the EMT model in BEAS-2B cells. Immunofluorescence and western blot were executed to verify the establishment of the EMT model. NEAT1, miR-204-5p, and Six1 expression levels were evaluated using RT-qPCR. The role of NEAT1 in EMT in vitro was explored by CCK8 assays and flow cytometry. The luciferase reporter assay was performed to validate the interaction between NEAT1 and miR-204-5p/Six1. RESULTS: NEAT1 expression was increased during EMT. Functional experiments showed that the knockdown of NEAT1 suppressed cell proliferation and promoted cell apoptosis in vitro. Furthermore, inhibition of NEAT1 decreased the expression of N-cadherin, vimentin, and α-SMA and increased the expression of E-cadherin. Mechanistically, NEAT1 was identified as a sponge for miR-204-5p, and Six1 was found to be a direct target of miR-204-5p. CONCLUSION: Down-regulation of NEAT1 reduced the Six1 expression via targeting miR-204-5p to inhibit the process of EMT in asthma. This study may provide new insight to reveal the underlying mechanisms of asthma.

Fisetin reduces ovalbumin-triggered airway remodeling by preventing phenotypic switching of airway smooth muscle cells.

BACKGROUND: The transformation of airway smooth muscle cells (ASMCs) from a quiescent phenotype to a hypersecretory and hypercontractile phenotype is a defining feature of asthmatic airway remodeling. Fisetin, a flavonoid compound, possesses anti-inflammatory characteristics in asthma; yet, its impact on airway remodeling and ASMCs phenotype transition has not been investigated. OBJECTIVES: This research seeked to assess the impact of fisetin on ovalbumin (OVA) induced asthmatic airway remodeling and ASMCs phenotype transition, and clarify the mechanisms through network pharmacology predictions as well as in vivo and in vitro validation. METHODS: First, a fisetin-asthma-ASMCs network was constructed to identify potential targets. Subsequently, cellular and animal studies were carried out to examine the inhibitory effects of fisetin on airway remodeling in asthmatic mice, and to detemine how fisetin impacts the phenotypic transition of ASMCs. RESULTS: Network analysis indicated that fisetin might affect asthma via mediating the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) pathway. Intraperitoneal administration of fisetin in vivo reduced airway inflammation and remodeling, as shown by reduced inflammatory cells, decreased T helper type 2 (Th2) cytokine release, diminished collagen accumulation, mitigated airway smooth muscle thickening, and decreased expression of osteopontin (OPN), collagen-I and α-smooth muscle actin (α-SMA). Moreover, fisetin suppressed the PI3K/AKT pathway in asthmatic lung tissue. According to the in vitro data, fisetin downregulated the expression of the synthetic phenotypic proteins OPN and collagen-I, contractile protein α-SMA, and inhibited cellular migration, potentially through the PI3K/AKT pathway. CONCLUSION: These results suggest that fisetin inhibits airway remodeling in asthma by regulating ASMCs phenotypic shift, emphasizing that fisetin is a promising candidate for the treatment of airway smooth muscle remodeling.

Distinctive CD39+CD9+ lung interstitial macrophages suppress IL-23/Th17-mediated neutrophilic asthma by inhibiting NETosis.

The IL-23-Th17 axis is responsible for neutrophilic inflammation in various inflammatory diseases. Here, we discover a potential pathway to inhibit neutrophilic asthma. In our neutrophil-dominant asthma (NDA) model, single-cell RNA-seq analysis identifies a subpopulation of CD39+CD9+ interstitial macrophages (IMs) suppressed by IL-23 in NDA conditions but increased by an IL-23 inhibitor αIL-23p19. Adoptively transferred CD39+CD9+ IMs suppress neutrophil extracellular trap formation (NETosis), a representative phenotype of NDA, and also Th17 cell activation and neutrophilic inflammation. CD39+CD9+ IMs first attach to neutrophils in a CD9-dependent manner, and then remove ATP near neutrophils that contribute to NETosis in a CD39-dependent manner. Transcriptomic data from asthmatic patients finally show decreased CD39+CD9+ IMs in severe asthma than mild/moderate asthma. Our results suggest that CD39+CD9+ IMs function as a potent negative regulator of neutrophilic inflammation by suppressing NETosis in the IL-23-Th17 axis and can thus serve as a potential therapeutic target for IL-23-Th17-mediated neutrophilic asthma.

Tezepelumab for severe asthma: elevating current practice to recognize epithelial driven profiles.

BACKGROUND: An increasing amount of evidence supports the relevance of epithelium across the wide spectrum of asthma pathobiology. On a clinical ground tezepelumab, selectively binding TSLP, a major epithelial cytokine, has demonstrated to be effective in asthma patients regardless their specific phenotype. In order to avoid the risk of considering tezepelumab as a not-specific option, the present perspective aims to sketch the tezepelumab best eligible patient profile and to propose some hallmarks of epithelial-driven disease by reviewing the published evidence on the drug mechanism of action and efficacy data. MAIN BODY: Although it cannot rely on standardised or exclusive "markers", the relationship between environment and poor asthma control might suggest a major relevance of the epithelial barrier dysfunction. In that light, allergy and asthma exacerbations concomitant with specific exposures (pathogens, pollutants, chemicals), as well as increased susceptibility to infections can be considered as the hallmark of an impaired epithelial immune response. Tezepelumab is effective in allergic patients, being able to reduce asthma exacerbations precipitated by the exposure to seasonal or perennial aeroallergens, including fungi. In addition, tezepelumab reduced the incidence of co-occurring respiratory illness and asthma exacerbations. In terms of inflammation, epithelial immune response has been related to an impaired mucus hypersecretion and plugging. A placebo-controlled trial demonstrated a significant reduction of mucus plugging in treated patient. Airways hyperreactivity (AHR), airways obstruction and remodelling have been described as an expression of epithelial orchestrated immunological activation. Of note, a significantly higher incidence of mannitol negative test in patients treated with tezepelumab when compared to placebo group has been observed. In addition, A 130 mL improvement in pre-BD FEV1 has been described in patients assuming Tezepelumab. The above-mentioned data suggest that bronchial reversibility and AHR can be considered "functional biomarkers" supporting patients' phenotyping and the identification of tezepelumab best responders. CONCLUSION: Integrating "functional biomarkers" to the inflammatory ones and a better characterization of asthma exacerbations might pave the way to a different and more transversal phenotyping, which overcomes the "restrictive" labels including T2 high, allergic/atopic or T2 low asthma. Precisely defining the disease characteristics and potential targets for a better control even in tezepelumab eligible subjects is essential to avoid the block buster temptation and optimize the personalized medicine approach according to each patient's individuality.

Metabolic and ionic control of T cells in asthma endotypes.

CD4+ T cells play a central role in orchestrating the immune response in asthma, with dysregulated ion channel profiles and altered metabolic signatures contributing to disease progression and severity. An important classification of asthma is based on the presence of T-helper cell type 2 (Th2) inflammation, dividing patients into Th2-high and Th2-low endotypes. These distinct endotypes have implications for disease severity, treatment response, and prognosis. By elucidating how ion channels and energy metabolism control Th cells in asthma, this review contributes to the pathophysiological understanding and the prospective development of personalized therapeutic treatment strategies for patients suffering from distinct asthma endotypes.

Release of sputum neutrophil granules is associated with pulmonary function and disease severity in childhood asthma.

BACKGROUND: Myeloperoxidase (MPO) and human neutrophil lipocalin or neutrophil gelatinase-associated lipocalin (HNL/NGAL) are stored in neutrophil granulocytes and secreted upon activation of the cells. They have been proposed to reflect the degree of inflammation in the airways. However, their role as potential markers of disease severity in childhood asthma remains unknown. This study investigated the relationship between the expression of MPO and HNL/NGAL and childhood asthma. METHODS: A total of 83 pediatric patients with asthma and 59 controls were enrolled. Using enzyme-linked immunosorbent assays, the human MPO and HNL/NGAL levels were measured in sputum supernatants. Assessments including spirometry, methacholine challenge test, and atopy test were conducted. RESULTS: No difference in sputum neutrophil counts was observed between pediatric patients with asthma and controls. However, sputum MPO and HNL/NGAL levels were significantly higher in patients with asthma than in controls (p = 0.021 and p < 0.001, respectively), especially in patients with moderate-to-severe persistent asthma. In patients with asthma, sputum MPO and HNL/NGAL levels showed a positive correlation with sputum neutrophil counts (MPO, r = 0.433, p < 0.001; HNL/NGAL, r = 0.584, p < 0.001) and with each other (r = 0.628, p < 0.001). Moreover, sputum HNL/NGAL level demonstrated better ability to accurately reflect current pulmonary function, airway inflammation, and limitations than MPO level in this study. CONCLUSIONS: Sputum MPO and HNL/NGAL levels, which reflect neutrophil activation in airways, were increased in pediatric patients with asthma. Moreover, sputum MPO and HNL/NGAL may serve as appropriate assessment indicators of asthma severity in pediatric patients.

TRIM11 Prevents Ferroptosis in model of asthma by UBE2N-TAX1BP1 signaling.

Asthma is a complex chronic respiratory inflammatory disease affected by both genetic and environmental factors. Therefore, our study explored the influence of TRIM11 on asthma and its underlying mechanisms. Our research involved patients diagnosed with asthma and healthy volunteers recruited from our hospital. We observed a reduction in serum TRIM11 expression in asthma patients, which positively correlated with the levels of anti-IgE or IgE. Additionally, both TRIM11 mRNA and protein expression in lung tissue were diminished. The introduction of the TRIM11 gene resulted in a reduction in inflammation in an in vitro asthma model and prevented the development of asthma in a mouse model. Moreover, the TRIM11 gene exhibited a suppressive effect on Ferroptosis and mitigated ROS-induced mitochondrial damage in the asthma model. TRIM11 was found to stimulate UBE2N-TAX1BP1 signaling in the asthma model, with UBE2N being identified as the specific target for TRIM11's effects on Ferroptosis. Furthermore, TRIM11 protein interacted with UBE2N protein and facilitated the dissociation of UBE2N-UBE2N in the asthma model. In conclusion, TRIM11 plays a vital role in preventing Ferroptosis in the asthma model through UBE2N-TAX1BP1 signaling. This indicates that targeting the TRIM11 mechanism could serve as a promising strategy for anti-Ferroptosis immunotherapy in asthma treatment.

Immunomodulatory Effect of Adipose Stem Cell-Derived Extra-Cellular Vesicles on Cytokine Expression and Regulatory T Cells in Patients with Asthma.

Although mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are as effective as MSCs in the suppression of allergic airway inflammation, few studies have evaluated the immunomodulatory capacity of MSC-derived EVs in patients with asthma. Thus, we assessed the effects of adipose stem cell (ASC)-derived EVs on cytokine expression and regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) of asthmatic patients. PBMCs (1 × 106 cells/mL) were isolated from asthmatic patient and healthy controls and co-cultured with 1 µg/mL of ASC-derived EVs. Th (T helper) 1-, Th2-, and Treg-related cytokine expression, fluorescence-activated cell sorting analysis of CD4+CD25+FOXP3+ T cells, and co-stimulatory molecules were analyzed before and after ASC-derived EV treatment. The expression levels of IL-4 and costimulatory molecules such as CD83 and CD86 were significantly higher in PBMCs of asthmatic patients than in control PBMCs. However, ASC-derived EV treatment significantly decreased the levels of interleukin (IL)-4 and co-stimulatory molecules such as CD83 and CD86 in the phytohemagglutinin (PHA)-stimulated PBMC of asthmatic patients. Furthermore, ASC-derived EVs remarkably increased the transforming growth factor-ß (TGF-ß) levels and expression of Tregs in the PBMC of asthmatic patients. ASC-derived EVs induce Treg expansion and have immunomodulatory effects by downregulating IL-4 and upregulating TGF-ß in PBMCs of asthmatic patients.

Betamethasone improved near-term neonatal lamb lung maturation in experimental maternal asthma.

Maternal asthma is associated with increased rates of neonatal lung disease, and fetuses from asthmatic ewes have fewer surfactant-producing cells and lower surfactant-protein B gene (SFTPB) expression than controls. Antenatal betamethasone increases lung surfactant production in preterm babies, and we therefore tested this therapy in experimental maternal asthma. Ewes were sensitised to house dust mite allergen, and an asthmatic phenotype induced by fortnightly allergen lung challenges; controls received saline. Pregnant asthmatic ewes were randomised to receive antenatal saline (asthma) or 12 mg intramuscular betamethasone (asthma+beta) at 138 and 139 days of gestation (term = 150 days). Lambs were delivered by Caesarean section at 140 days of gestation and ventilated for 45 min before tissue collection. Lung function and structure were similar in control lambs (n = 16, 11 ewes) and lambs from asthma ewes (n = 14, 9 ewes). Dynamic lung compliance was higher in lambs from asthma+beta ewes (n = 12, 8 ewes) compared to those from controls (P = 0.003) or asthma ewes (P = 0.008). Lung expression of surfactant protein genes SFTPA (P = 0.048) and SFTPB (P < 0.001), but not SFTPC (P = 0.177) or SFTPD (P = 0.285), was higher in lambs from asthma+beta than those from asthma ewes. Female lambs had higher tidal volume (P = 0.007), dynamic lung compliance (P < 0.001), and SFTPA (P = 0.037) and SFTPB gene expression (P = 0.030) than males. These data suggest that betamethasone stimulates lung maturation and function of near-term neonates, even in the absence of impairment by maternal asthma.

Kiss1 receptor knockout exacerbates airway hyperresponsiveness and remodeling in a mouse model of allergic asthma.

BACKGROUND: In asthma, sex-steroids signaling is recognized as a critical regulator of disease pathophysiology. However, the paradoxical role of sex-steroids, especially estrogen, suggests that an upstream mechanism or even independent of estrogen plays an important role in regulating asthma pathophysiology. In this context, in our previous studies, we explored kisspeptin (Kp) and its receptor Kiss1R's signaling in regulating human airway smooth muscle cell remodeling in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the effect of endogenous Kp in regulating AHR and remodeling using Kiss1R knockout (Kiss1R-/-) mice. METHODS: C57BL/6J WT (Kiss1R+/+) and Kiss1R-/- mice, both male and female, were intranasally challenged with mixed-allergen (MA) and/or phosphate-buffered saline (PBS). We used flexiVent analysis to assess airway resistance (Rrs), elastance (Ers), and compliance (Crs). Following this, broncho-alveolar lavage (BAL) was performed for differential leukocyte count (DLC) and cytokine analysis. Histology staining was performed using hematoxylin and eosin (H&E) for morphological analysis and Masson's Trichrome (MT) for collagen deposition. Additionally, lung sections were processed for immunofluorescence (IF) of Ki-67, α-smooth muscle actin (α-SMA), and tenascin-c. RESULTS: Interestingly, the loss of Kiss1R exacerbated lung function and airway contractility in mice challenged with MA, with more profound effects in Kiss1R-/- female mice. MA-challenged Kiss1R-/- mice showed a significant increase in immune cell infiltration and proinflammatory cytokine levels. Importantly, the loss of Kiss1R aggravated Th2/Th17 biased cytokines in MA-challenged mice. Furthermore, histology of lung sections from Kiss1R-/- mice showed increased collagen deposition on airway walls and mucin production in airway cells compared to Kiss1R+/+ mice. In addition, immunofluorescence analysis showed loss of Kiss1R significantly aggravated airway remodeling and subsequently AHR. CONCLUSIONS: These findings demonstrate the importance of inherent Kiss1R signaling in regulating airway inflammation, AHR, and remodeling in the pathophysiology of asthma.

Single-Cell Hypertrophy Promotes Contractile Function of Cultured Human Airway Smooth Muscle Cells via Piezo1 and YAP Auto-Regulation.

Severe asthma is characterized by increased cell volume (hypertrophy) and enhanced contractile function (hyperresponsiveness) of the airway smooth muscle cells (ASMCs). The causative relationship and underlying regulatory mechanisms between them, however, have remained unclear. Here, we manipulated the single-cell volume of in vitro cultured human ASMCs to increase from 2.7 to 5.2 and 8.2 × 103 µm3 as a simulated ASMC hypertrophy by culturing the cells on micropatterned rectangular substrates with a width of 25 µm and length from 50 to 100 and 200 µm, respectively. We found that as the cell volume increased, ASMCs exhibited a pro-contractile function with increased mRNA expression of contractile proteins, increased cell stiffness and traction force, and enhanced response to contractile stimulation. We also uncovered a concomitant increase in membrane tension and Piezo1 mRNA expression with increasing cell volume. Perhaps more importantly, we found that the enhanced contractile function due to cell volume increase was largely attenuated when membrane tension and Piezo1 mRNA expression were downregulated, and an auto-regulatory loop between Piezo1 and YAP mRNA expression was also involved in perpetuating the contractile function. These findings, thus, provide convincing evidence of a direct link between hypertrophy and enhanced contractile function of ASMCs that was mediated via Piezo1 mRNA expression, which may be specifically targeted as a novel therapeutic strategy to treat pulmonary diseases associated with ASMC hypertrophy such as severe asthma.