scRNA-seq identifies unique macrophage population in murine model of ozone induced asthma exacerbation.

Ozone (O 3 ) inhalation triggers asthmatic airway hyperresponsiveness (AHR), but the mechanisms by which this occurs are unknown. Previously, we developed a murine model of dust mite, ragweed, and aspergillus (DRA)-induced allergic lung inflammation followed by O 3 exposure for mechanistic investigation. The present study used single cell RNA-sequencing for unbiased profiling of immune cells within the lungs of mice exposed to DRA, O 3 , or DRA+O 3 , to identify the components of the immune cell niche that contribute to AHR. Alveolar macrophages (AMs) had the greatest number of differentially expressed genes following DRA+O 3 , most of which were unique to the 2-hit exposure. Following DRA+O 3 , AMs activated transcriptional pathways related to cholesterol biosynthesis, degradation of the extracellular matrix, endosomal TLR processing, and various cytokine signals. We also identified AM and monocyte subset populations that were unique to the DRA+O 3 group. These unique AMs activated gene pathways related to inflammation, sphingolipid metabolism, and bronchial constriction. The unique monocyte population had a gene signature that suggested phospholipase activation and increased degradation of the extracellular matrix. Flow cytometry analysis of BAL immune cells showed recruited monocyte-derived AMs after DRA and DRA+O 3 , but not after O 3 exposure alone. O 3 alone increased BAL neutrophils but this response was attenuated in DRA+O 3 mice. DRA-induced changes in the airspace immune cell profile were reflected in elevated BAL cytokine/chemokine levels following DRA+O 3 compared to O 3 alone. The present work highlights the role of monocytes and AMs in the response to O 3 and suggests that the presence of distinct subpopulations following allergic inflammation may contribute to O 3 -induced AHR.

Innate immune responses are increased in children with acute asthma exacerbation.

BACKGROUND: Little is known about the immune responses during acute asthma exacerbation. In this study, we examined immune responses in children following an acute asthma exacerbation. METHODS: We evaluated pro-inflammatory cytokine levels and gene expression profiles in blood samples from pediatric patients admitted for acute asthma exacerbation. Viral PCR was performed to differentiate between viral or non-viral-associated exacerbations. RESULTS: Following informed consent, clinical data were obtained from 20 children with asthma (median [interquartile range, IQR]: age 11.5 [8.0, 14.2]) years and 14 healthy age-matched controls (10.5 [7.0, 13.0]). Twelve had positive nasopharyngeal Polymerase chain reaction (PCR) for viral infection (11 rhinoviruses and 1 respiratory syncytial virus (RSV)). Nine were in the pediatric intensive care unit (PICU) and among them five required continuous positive airway pressure (CPAP). Mean (±SD) days on systemic steroids before drawing blood sample were 2.5 ± 1.6. Twelve had history of environmental allergies with 917 (274, 1396) IU/mL total IgE (median (IQR)). Compared with controls, IL-1RA and IL-10 levels were significantly increased and TNF-α significantly decreased in asthma subjects (p < .05 for all). RNA-seq analysis revealed 852 differentially expressed genes in subjects with asthma. Pathway analysis found upregulated genes and pathways involved in innate immune responses in subjects with asthma. Significantly reduced genes included pathways associated with T helper cell differentiation and activation. CONCLUSIONS: In acute asthma exacerbation, innate immune pathways remained increased while adaptive immune responses related to T helper cells are blunted and are independent of trigger or asthma severity. Our novel findings highlight the need to identify new therapies to target persistent innate immune responses to improve outcomes in acute asthma.

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Predictors of recurrent wheezing in late preterm infants.

INTRODUCTION: Premature infants have an increased risk of respiratory morbidity, including the development of recurrent wheezing. We sought to determine perinatal factors in late preterm infants associated with an increased risk of recurrent wheezing in the first 3 years of life. METHODS: A retrospective chart review of infants born between 32 and 36 weeks gestational age at a tertiary hospital from 2013 to 2016 was performed. Infants with any co-morbid medical conditions were excluded. Recurrent wheezing was identified by two or more visit diagnoses for reactive airway disease, wheezing-associated respiratory infection, wheezing, or asthma during the first 3 years of life. Those with recurrent wheezing were compared to matched preterm infants who did not develop wheezing. RESULTS: Three hundred and fourteen late preterm infants were included in this study; 210 infants developed recurrent wheezing while 104 did not. Gender, sex, and race were comparable between both groups. Development of wheezing was associated with positive family history of asthma (p = .014), receiving antibiotics during the neonatal period (p < .001), requiring continuous positive airway pressure for <24 h (p = .019), and receiving supplemental oxygen during the newborn period (p = .023). CONCLUSION: This study retrospectively identified risk factors associated with development of wheezing in late preterm infants. Prospective studies are needed to determine whether these factors will predict recurrent wheeze in this patient population.

Macrophages Orchestrate Airway Inflammation, Remodeling, and Resolution in Asthma.

Asthma is a heterogenous chronic inflammatory lung disease with endotypes that manifest different immune system profiles, severity, and responses to current therapies. Regardless of endotype, asthma features increased immune cell infiltration, inflammatory cytokine release, and airway remodeling. Lung macrophages are also heterogenous in that there are separate subsets and, depending on the environment, different effector functions. Lung macrophages are important in recruitment of immune cells such as eosinophils, neutrophils, and monocytes that enhance allergic inflammation and initiate T helper cell responses. Persistent lung remodeling including mucus hypersecretion, increased airway smooth muscle mass, and airway fibrosis contributes to progressive lung function decline that is insensitive to current asthma treatments. Macrophages secrete inflammatory mediators that induce airway inflammation and remodeling. Additionally, lung macrophages are instrumental in protecting against pathogens and play a critical role in resolution of inflammation and return to homeostasis. This review summarizes current literature detailing the roles and existing knowledge gaps for macrophages as key inflammatory orchestrators in asthma pathogenesis. We also raise the idea that modulating inflammatory responses in lung macrophages is important for alleviating asthma.

Sterols and immune mechanisms in asthma.

The field of sterol and oxysterol biology in lung disease has recently gained attention, revealing a unique need for sterol uptake and metabolism in the lung. The presence of cholesterol transport, biosynthesis, and sterol/oxysterol-mediated signaling in immune cells suggests a role in immune regulation. In support of this idea, statin drugs that inhibit the cholesterol biosynthesis rate-limiting step enzyme, hydroxymethyl glutaryl coenzyme A reductase, show immunomodulatory activity in several models of inflammation. Studies in human asthma reveal contradicting results, whereas promising retrospective studies suggest benefits of statins in severe asthma. Here, we provide a timely review by discussing the role of sterols in immune responses in asthma, analytical tools to evaluate the role of sterols in disease, and potential mechanistic pathways and targets relevant to asthma. Our review reveals the importance of sterols in immune processes and highlights the need for further research to solve critical gaps in the field.

Aging-Related Mechanisms Contribute to Corticosteroid Insensitivity in Elderly Asthma.

Asthma in elderly populations is an increasing health problem that is accompanied by diminished lung function and frequent exacerbations. As potent anti-inflammatory drugs, corticosteroids are commonly used to reduce lung inflammation, improve lung function, and manage disease symptoms in asthma. Although effective for most individuals, older patients are more insensitive to corticosteroids, making it difficult to manage asthma in this population. With the number of individuals older than 65 continuing to increase, it is important to understand the distinct mechanisms that promote corticosteroid insensitivity in the aging lung. In this review, we discuss corticosteroid insensitivity in asthma with an emphasis on mechanisms that contribute to persistent inflammation and diminished lung function in older individuals.

Impact of elexacaftor-tezacaftor-ivacaftor on bacterial colonization and inflammatory responses in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a multisystem disease with progressive deterioration. Recently, CF transmembrane conductance regulator (CFTR) modulator therapies were introduced that repair underlying protein defects. Objective of this study was to determine the impact of elexacaftor-tezacaftor-ivacaftor (ETI) on clinical parameters and inflammatory responses in people with CF (pwCF). METHODS: Lung function (FEV1 ), body mass index (BMI) and microbiologic data were collected at initiation and 3-month intervals for 1 year. Blood was analyzed at baseline and 6 months for cytokines and immune cell populations via flow cytometry and compared to non-CF controls. RESULTS: Sample size was 48 pwCF, 28 (58.3%) males with a mean age of 28.8 ± 10.7 years. Significant increases in %predicted FEV1 and BMI were observed through 6 months of ETI therapy with no change thereafter. Changes in FEV1 and BMI at 3 months were significantly correlated (r = 57.2, p < 0.01). There were significant reductions in Pseudomonas and Staphylococcus positivity (percent of total samples) in pwCF through 12 months of ETI treatment. Healthy controls (n = 20) had significantly lower levels of circulating neutrophils, interleukin (IL)-6, IL-8, and IL-17A and higher levels of IL-13 compared to pwCF at baseline (n = 48). After 6 months of ETI, pwCF had significant decreases in IL-8, IL-6, and IL-17A levels and normalization of peripheral blood immune cell composition. CONCLUSIONS: In pwCF, ETI significantly improved clinical outcomes, reduced systemic pro-inflammatory cytokines, and restored circulating immune cell composition after 6 months of therapy.

Sterols in asthma.

While sterols regulate immune processes key to the pathogenesis of asthma, inhibition of sterols with statin drugs has shown conflicting results in human asthma. Here, a novel understanding of the impact of sterols on type 17 immune responses and asthma lead us to hypothesize that sterols and statins may be relevant to severe asthma endotypes with neutrophil infiltration.

Th1 cytokines synergize to change gene expression and promote corticosteroid insensitivity in pediatric airway smooth muscle.

BACKGROUND: Corticosteroids remain a key therapy for treating children with asthma. Patients with severe asthma are insensitive, resistant, or refractory to corticosteroids and have poorly controlled symptoms that involve airway inflammation, airflow obstruction, and frequent exacerbations. While the pathways that mediate corticosteroid insensitivity in asthma remain poorly defined, recent studies suggest that enhanced Th1 pathways, mediated by TNFα and IFNγ, may play a role. We previously reported that the combined effects of TNFα and IFNγ promote corticosteroid insensitivity in developing human airway smooth muscle (ASM). METHODS: To further understand the effects of TNFα and IFNγ on corticosteroid sensitivity in the context of neonatal and pediatric asthma, we performed RNA sequencing (RNA-seq) on human pediatric ASM treated with fluticasone propionate (FP), TNFα, and/or IFNγ. RESULTS: We found that TNFα had a greater effect on gene expression (~ 1000 differentially expressed genes) than IFNγ (~ 500 differentially expressed genes). Pathway and transcription factor analyses revealed enrichment of several pro-inflammatory responses and signaling pathways. Interestingly, treatment with TNFα and IFNγ augmented gene expression with more than 4000 differentially expressed genes. Effects of TNFα and IFNγ enhanced several pro-inflammatory genes and pathways related to ASM and its contributions to asthma pathogenesis, which persisted in the presence of corticosteroids. Co-expression analysis revealed several gene networks related to TNFα- and IFNγ-mediated signaling, pro-inflammatory mediator production, and smooth muscle contractility. Many of the co-expression network hubs were associated with genes that are insensitive to corticosteroids. CONCLUSIONS: Together, these novel studies show the combined effects of TNFα and IFNγ on pediatric ASM and implicate Th1-associated cytokines in promoting ASM inflammation and hypercontractility in severe asthma.

Chronic Allergen Challenge Induces Corticosteroid Insensitivity With Persistent Airway Remodeling and Type 2 Inflammation.

Type 2-high severe asthma is described as a distinct endotype with Th2 inflammation, high eosinophil lung infiltration, impaired lung function, and reduced corticosteroid sensitivity. While the inflammatory milieu is similar to mild asthma, patients with type 2-high severe asthma likely have underlying mechanisms that sustain asthma pathophysiology despite corticosteroid treatments. Acute and chronic allergen models induce robust type 2 inflammatory responses, however differences in corticosteroid sensitivity remains poorly understood. In the present study, we sensitized and challenged mice with ovalbumin (OVA; acute model) or mixed allergens (MA; chronic model). Corticosteroid sensitivity was assessed by administering vehicle, 1, or 3 mg/kg fluticasone propionate (FP) and examining key asthmatic features such as airway inflammation, remodeling, hyperresponsiveness, and antioxidant capacity. Both acute and chronic allergen exposure exhibited enhanced AHR, immune cell infiltration, airway inflammation, and remodeling, but corticosteroids were unable to fully alleviate inflammation, AHR, and airway smooth muscle mass in MA-challenged mice. While there were no differences in antioxidant capacity, persistent IL-4+ Th2 cell population suggests the MA model induces type 2 inflammation that is insensitive to corticosteroids. Our data indicate that chronic allergen exposure is associated with more persistent type 2 immune responses and corticosteroid insensitivity. Understanding differences between acute and chronic allergen models could unlock underlying mechanisms related to type 2-high severe asthma.

PRMT5 in T Cells Drives Th17 Responses, Mixed Granulocytic Inflammation, and Severe Allergic Airway Inflammation.

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt's agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

Corticosteroid insensitivity persists in the absence of STAT1 signaling in severe allergic airway inflammation.

Corticosteroid insensitivity in asthma limits the ability to effectively manage severe asthma, which is characterized by persistent airway inflammation, airway hyperresponsiveness (AHR), and airflow obstruction despite corticosteroid treatment. Recent reports indicate that corticosteroid insensitivity is associated with increased interferon-γ (IFN-γ) levels and T-helper (Th) 1 lymphocyte infiltration in severe asthma. Signal transducer and activator of transcription 1 (STAT1) activation by IFN-γ is a key signaling pathway in Th1 inflammation; however, its role in the context of severe allergic airway inflammation and corticosteroid sensitivity remains unclear. In this study, we challenged wild-type (WT) and Stat1-/- mice with mixed allergens (MA) augmented with c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate], an inducer of Th1 cell infiltration with increased eosinophils, neutrophils, Th1, Th2, and Th17 cells. Compared with WT mice, Stat1-/- had reduced neutrophils, Th1, and Th17 cell infiltration. To evaluate corticosteroid sensitivity, mice were treated with either vehicle, 1 or 3 mg/kg fluticasone propionate (FP). Corticosteroids significantly reduced eosinophil infiltration and cytokine levels in both c-di-GMP + MA-challenged WT and Stat1-/- mice. However, histological and functional analyses show that corticosteroids did not reduce airway inflammation, epithelial mucous cell abundance, airway smooth muscle mass, and AHR in c-di-GMP + MA-challenged WT or Stat1-/- mice. Collectively, our data suggest that increased Th1 inflammation is associated with a decrease in corticosteroid sensitivity. However, increased airway pathology and AHR persist in the absence of STAT1 indicate corticosteroid insensitivity in structural airway cells is a STAT1 independent process.

Oxidative Stress Promotes Corticosteroid Insensitivity in Asthma and COPD.

Corticosteroid insensitivity is a key characteristic of patients with severe asthma and COPD. These individuals experience greater pulmonary oxidative stress and inflammation, which contribute to diminished lung function and frequent exacerbations despite the often and prolonged use of systemic, high dose corticosteroids. Reactive oxygen and nitrogen species (RONS) promote corticosteroid insensitivity by disrupting glucocorticoid receptor (GR) signaling, leading to the sustained activation of pro-inflammatory pathways in immune and airway structural cells. Studies in asthma and COPD models suggest that corticosteroids need a balanced redox environment to be effective and to reduce airway inflammation. In this review, we discuss how oxidative stress contributes to corticosteroid insensitivity and the importance of optimizing endogenous antioxidant responses to enhance corticosteroid sensitivity. Future studies should aim to identify how antioxidant-based therapies can complement corticosteroids to reduce the need for prolonged high dose regimens in patients with severe asthma and COPD.

Impact of guideline adherence and race on asthma control in children.

BACKGROUND: Asthma control in African Americans (AA) is considered more difficult to achieve than in Caucasian Americans (CA). The aim of this study was to compare asthma control over time among AA and CA children whose asthma is managed per NAEPP (EPR-3) guidelines. METHODS: This was a one-year prospective study of children referred by their primary care physicians for better asthma care in a specialty asthma clinic. All children received asthma care per NAEPP guidelines. Results were compared between CA and AA children at baseline and then at three-month intervals for one year. RESULTS: Of the 345 children, ages 2-17 years (mean = 6.2 ± 4), 220 (63.8%) were CA and 125 (36.2%) were AA. There were no significant differences in demographics other than greater pet ownership in CA families. At baseline, AA children had significantly more visits to the Emergency Department for acute asthma symptoms (mean = 2.3 [Formula: see text] compared to CA (1.4 ± 2.3, P = 0.003). There were no other significant differences in acute care utilization, asthma symptoms (mean days/month), or mean asthma control test (ACT) scores at baseline. Within 3-6 months, in both groups, mean ACT scores, asthma symptoms and acute care utilization significantly improved (P < 0.05 for all) and change over time in both groups was comparable except for a significantly greater decrease in ED visits in AA children compared to CA children (P = 002). CONCLUSION: Overall, improvement in asthma control during longitudinal assessment was similar between AA and CA children because of  consistent use of NAEPP asthma care guidelines.

Network and co-expression analysis of airway smooth muscle cell transcriptome delineates potential gene signatures in asthma.

Airway smooth muscle (ASM) is known for its role in asthma exacerbations characterized by acute bronchoconstriction and remodeling. The molecular mechanisms underlying multiple gene interactions regulating gene expression in asthma remain elusive. Herein, we explored the regulatory relationship between ASM genes to uncover the putative mechanism underlying asthma in humans. To this end, the gene expression from human ASM was measured with RNA-Seq in non-asthmatic and asthmatic groups. The gene network for the asthmatic and non-asthmatic group was constructed by prioritizing differentially expressed genes (DEGs) (121) and transcription factors (TFs) (116). Furthermore, we identified differentially connected or co-expressed genes in each group. The asthmatic group showed a loss of gene connectivity due to the rewiring of major regulators. Notably, TFs such as ZNF792, SMAD1, and SMAD7 were differentially correlated in the asthmatic ASM. Additionally, the DEGs, TFs, and differentially connected genes over-represented in the pathways involved with herpes simplex virus infection, Hippo and TGF-ß signaling, adherens junctions, gap junctions, and ferroptosis. The rewiring of major regulators unveiled in this study likely modulates the expression of gene-targets as an adaptive response to asthma. These multiple gene interactions pointed out novel targets and pathways for asthma exacerbations.

Selective YAP/TAZ inhibition in fibroblasts via dopamine receptor D1 agonism reverses fibrosis.

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.

Early Life Represents a Vulnerable Time Window for IL-33-Induced Peripheral Lung Pathology.

IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33-mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.