Optimising the management of children with concomitant bladder dysfunction and behavioural disorders.

Bladder dysfunction and behavioural disorders in children are commonly concomitant; hence, it is difficult to treat each in isolation. Pharmacotherapy is common treatment for behavioural disorders, and these medications may have intended or unintended positive or negative bladder sequelae. This review identifies the literature regarding the effects of behavioural pharmacotherapy on bladder functioning and possible bladder management strategies in children with concomitant behaviour and bladder disorders to enable clinicians to better manage both conditions. A PROSPERO registered PRISMA-guided review of three major databases was performed. After an initial scoping study revealed significant heterogeneity, a narrative approach was undertaken to discuss the results of all relevant cases relating to children being treated with pharmacotherapy for behaviour disorders and outcomes related to bladder function. Studies were screened to identify those that described effects of commonly prescribed medications in children with behavioural disorders such as stimulants, alpha 2 agonists, tricyclic antidepressants (TCA), serotonin and noradrenergic reuptake inhibitors (SNRI), selective serotonin reuptake inhibitors (SSRI) and antipsychotics, and the findings and implications were summarised. The review identified 46 studies relevant to behavioural pharmacotherapy and bladder function (stimulants (n = 9), alpha 2 agonists (n = 2), TCAs (n = 7), SNRIs (n = 8), SSRIs (n = 8) and antipsychotics (n = 6). Six studies focused specifically on bladder management in children with behavioural disorders with concurrent behavioural pharmacotherapy. This review identifies useful factors that may assist clinicians with predicting unintended bladder effects following initiation of behavioural pharmacotherapy to facilitate the best approach to the treatment of bladder dysfunction in children with behavioural disorders. With this evidence, we have provided a useful decision-making algorithm to aide clinicians in the management of these dual pathologies.

Airway and parenchymal transcriptomics in a novel model of asthma and COPD overlap.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) are common chronic respiratory diseases, and some patients have overlapping disease features, termed asthma-COPD overlap (ACO). Patients characterized with ACO have increased disease severity; however, the mechanisms driving this have not been widely studied. OBJECTIVES: This study sought to characterize the phenotypic and transcriptomic features of experimental ACO in mice induced by chronic house dust mite antigen and cigarette smoke exposure. METHODS: Female BALB/c mice were chronically exposed to house dust mite antigen for 11 weeks to induce experimental asthma, cigarette smoke for 8 weeks to induce experimental COPD, or both concurrently to induce experimental ACO. Lung inflammation, structural changes, and lung function were assessed. RNA-sequencing was performed on separated airway and parenchyma lung tissues to assess transcriptional changes. Validation of a novel upstream driver SPI1 in experimental ACO was assessed using the pharmacological SPI1 inhibitor, DB2313. RESULTS: Experimental ACO recapitulated features of both asthma and COPD, with mixed pulmonary eosinophilic/neutrophilic inflammation, small airway collagen deposition, and increased airway hyperresponsiveness. Transcriptomic analysis identified common and distinct dysregulated gene clusters in airway and parenchyma samples in experimental asthma, COPD, and ACO. Upstream driver analysis revealed increased expression of the transcription factor Spi1. Pharmacological inhibition of SPI1 using DB2313, reduced airway remodeling and airway hyperresponsiveness in experimental ACO. CONCLUSIONS: A new experimental model of ACO featuring chronic dual exposures to house dust mite and cigarette smoke mimics key disease features observed in patients with ACO and revealed novel disease mechanisms, including upregulation of SPI1, that are amenable to therapy.

Relationship between type 2 cytokine and inflammasome responses in obesity-associated asthma.

BACKGROUND: Obesity is a risk factor for asthma, and obese asthmatic individuals are more likely to have severe, steroid-insensitive disease. How obesity affects the pathogenesis and severity of asthma is poorly understood. Roles for increased inflammasome-mediated neutrophilic responses, type 2 immunity, and eosinophilic inflammation have been described. OBJECTIVE: We investigated how obesity affects the pathogenesis and severity of asthma and identified effective therapies for obesity-associated disease. METHODS: We assessed associations between body mass index and inflammasome responses with type 2 (T2) immune responses in the sputum of 25 subjects with asthma. Functional roles for NLR family, pyrin domain-containing (NLRP) 3 inflammasome and T2 cytokine responses in driving key features of disease were examined in experimental high-fat diet-induced obesity and asthma. RESULTS: Body mass index and inflammasome responses positively correlated with increased IL-5 and IL-13 expression as well as C-C chemokine receptor type 3 expression in the sputum of subjects with asthma. High-fat diet-induced obesity resulted in steroid-insensitive airway hyperresponsiveness in both the presence and absence of experimental asthma. High-fat diet-induced obesity was also associated with increased NLRP3 inflammasome responses and eosinophilic inflammation in airway tissue, but not lumen, in experimental asthma. Inhibition of NLRP3 inflammasome responses reduced steroid-insensitive airway hyperresponsiveness but had no effect on IL-5 or IL-13 responses in experimental asthma. Depletion of IL-5 and IL-13 reduced obesity-induced NLRP3 inflammasome responses and steroid-insensitive airway hyperresponsiveness in experimental asthma. CONCLUSION: We found a relationship between T2 cytokine and NLRP3 inflammasome responses in obesity-associated asthma, highlighting the potential utility of T2 cytokine-targeted biologics and inflammasome inhibitors.

Differences in pulmonary group 2 innate lymphoid cells are dependent on mouse age, sex and strain.

Innate lymphoid cells (ILCs) are resident in the lung and are involved in both the maintenance of homeostasis and the pathogenesis of respiratory diseases. In this study, murine lung ILCs were characterized using flow cytometry and the impact of mouse age, sex and strain were assessed. Lung ILCs were found as early as postnatal day 4 and numbers peaked at 2 weeks, and then decreased as the lung matured. During postnatal lung development, ILC expressed differential amounts of group 2 ILC (ILC2)-associated cell surface antigens including ST2, CD90.2 and ICOS. Using Il5venus Il13td-tomato dual reporter mice, neonates were found to have increased constitutive interleukin (IL)-13 expression compared with adult mice. Neonates and adults had similar ratios of IL-5+ CD45+ leukocytes; however, these cells were mostly composed of ILCs in neonates and T cells in adults. Sex-specific differences in ILC numbers were also observed, with females having greater numbers of lung ILCs than males in both neonatal and adult mice. Female lung ILCs also expressed higher levels of ICOS and decreased KLRG1. Mouse strain also impacted on lung ILCs with BALB/c mice having more ILCs in the lung and increased expression of ST2 and ICOS compared with C57BL/6J mice. Collectively, these data show that lung ILC numbers, cell surface antigen expression, IL-5 and IL-13 levels differed between neonatal and adult lung ILCs. In addition, cell surface antigens commonly used for ILC2 quantification, such as ST2, CD90.2 and ICOS, differ depending on age, sex and strain and these are important considerations for consistent universal identification of lung ILC2s.

The effects of increasing fruit and vegetable intake in children with asthma: A randomized controlled trial.

BACKGROUND: A high fruit and vegetable (F&V) diet reduces asthma exacerbations in adults; this has not been examined in children to date. OBJECTIVE: To investigate the effect of a 6-month, high F&V diet on the time to first asthma exacerbation in children with asthma, in a parallel-group, randomized, controlled trial. METHODS: Children (aged 3-11 years) with asthma, history of exacerbations and usual low F&V intake (≤3 serves/day) were randomized to the intervention (high F&V diet) or control group (usual diet) for 6 months. The primary outcome was time to first exacerbation requiring medical intervention. Secondary outcomes included exacerbation rate, lung function, plasma TNF-α, CRP, and IL-6, faecal microbiota and peripheral blood mononuclear cell (PBMC) histone deacetylase (HDAC) activity and G-protein coupled receptor (GPR) 41/43 and HDAC (1-11) expression. RESULTS: 67 children were randomized between September 2015 and July 2018. F&V intake (difference in change (∆): 3.5 serves/day, 95% CI: [2.6, 4.4] p < 0.001) and plasma total carotenoids (∆: 0.44 µg/ml [0.19, 0.70] p = 0.001) increased after 6 months (intervention vs control). Time to first exacerbation (HR: 0.81, 95% CI: [0.38, 1.69], p = 0.569; control vs. intervention) and exacerbation rate (IRR: 0.84, [0.47, 1.49], p = 0.553; control vs. intervention) were similar between groups. In per-protocol analysis, airway reactance z-scores increased in the intervention versus control group (X5 ∆: 0.76 [0.04, 1.48] p = 0.038, X20 ∆: 0.93 [0.23, 1.64] p = 0.009) and changes in faecal microbiota were observed though there was no difference between groups in systemic inflammation or molecular mechanisms. In the control group, CRP and HDAC enzyme activity increased, while GPR41 expression decreased. No adverse events attributable to the interventions were observed. CONCLUSION & CLINICAL RELEVANCE: A high F&V diet did not affect asthma exacerbations over the 6-month intervention, though warrants further investigation as a strategy for improving lung function and protecting against systemic inflammation in children with asthma.

Increased burden of rare variants in genes of the endosomal Toll-like receptor pathway in patients with systemic lupus erythematosus.

OBJECTIVE: To compare the frequency of rare variants in genes of the pathophysiologically relevant endosomal Toll-like receptor (eTLR) pathway and any quantifiable differences in variant rarity, predicted deleteriousness, or molecular proximity in patients with systemic lupus erythematosus (SLE) and healthy controls. PATIENTS AND METHODS: 65 genes associated with the eTLR pathway were identified by literature search and pathway analysis. Using next generation sequencing techniques, these were compared in two randomised cohorts of patients with SLE (n = 114 and n = 113) with 197 healthy controls. Genetically determined ethnicity was used to normalise minor allele frequencies (MAF) for the identified genetic variants and these were then compared by their frequency: rare (MAF < 0.005), uncommon (MAF 0.005-0.02), and common (MAF >0.02). This was compared to the results for 65 randomly selected genes. RESULTS: Patients with SLE are more likely to carry a rare nonsynonymous variant affecting proteins within the eTLR pathway than healthy controls. Furthermore, individuals with SLE are more likely to have multiple rare variants in this pathway. There were no differences in rarity, Combined Annotation Dependent Depletion (CADD) score, or molecular proximity for rare eTLR pathway variants. CONCLUSIONS: Rare non-synonymous variants are enriched in patients with SLE in the eTLR pathway. This supports the hypothesis that SLE arises from several rare variants of relatively large effect rather than many common variants of small effect.

Bronchioalveolar stem cells in lung repair, regeneration and disease.

Bronchioalveolar stem cells (BASCs) are a lung resident stem cell population located at bronchioalveolar duct junctions that contribute to the maintenance of bronchiolar club cells and alveolar epithelial cells of the distal lung. Their transformed counterparts are considered to be likely progenitors of lung adenocarcinomas, which has been a major area of research in relation to BASCs. A critical limitation in addressing the function of BASCs in vivo has been the lack of a unique BASC marker, which has prevented specific targeting of BASCs in animal models of respiratory conditions. Recently, there have been several studies describing genetically modified mice that allow in vivo quantification, tracing, and functional analysis of BASCs to address this long-standing issue. These cutting-edge experimental tools will likely have significant implications for future experimental studies involving BASCs and the elucidation of their role in various lung diseases. To date, this has been largely explored in models of lung injury including naphthalene-induced airway injury, bleomycin-induced alveolar injury, hyperoxia-induced models of bronchopulmonary dysplasia, and influenza virus infection. These novel experimental mouse tools will facilitate the assessment of the impact of BASC loss on additional respiratory conditions including infection-induced severe asthma and chronic obstructive pulmonary disease, as well as respiratory bacterial infections, both in early life and adulthood. These future studies may shed light on the potential broad applicability of targeting BASCs for a diverse range of respiratory conditions during lung development and in promoting effective regeneration and repair of the lung in respiratory diseases. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mechanisms and treatments for severe, steroid-resistant allergic airway disease and asthma.

Severe, steroid-resistant asthma is clinically and economically important since affected individuals do not respond to mainstay corticosteroid treatments for asthma. Patients with this disease experience more frequent exacerbations of asthma, are more likely to be hospitalized, and have a poorer quality of life. Effective therapies are urgently required, however, their development has been hampered by a lack of understanding of the pathological processes that underpin disease. A major obstacle to understanding the processes that drive severe, steroid-resistant asthma is that the several endotypes of the disease have been described that are characterized by different inflammatory and immunological phenotypes. This heterogeneity makes pinpointing processes that drive disease difficult in humans. Clinical studies strongly associate specific respiratory infections with severe, steroid-resistant asthma. In this review, we discuss key findings from our studies where we describe the development of representative experimental models to improve our understanding of the links between infection and severe, steroid-resistant forms of this disease. We also discuss their use in elucidating the mechanisms, and their potential for developing effective therapeutic strategies, for severe, steroid-resistant asthma. Finally, we highlight how the immune mechanisms and therapeutic targets we have identified may be applicable to obesity-or pollution-associated asthma.

Crucial role for lung iron level and regulation in the pathogenesis and severity of asthma.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.

Lung development and emerging roles for type 2 immunity.

Lung development is a complex process mediated through the interaction of multiple cell types, factors and mediators. In mice, it starts as early as embryonic day 9 and continues into early adulthood. The process can be separated into five different developmental stages: embryonic, pseudoglandular, canalicular, saccular, and alveolar. Whilst lung bud formation and branching morphogenesis have been studied extensively, the mechanisms of alveolarisation are incompletely understood. Aberrant lung development can lead to deleterious consequences for respiratory health such as bronchopulmonary dysplasia (BPD), a disease primarily affecting preterm neonates, which is characterised by increased pulmonary inflammation and disturbed alveolarisation. While the deleterious effects of type 1-mediated inflammatory responses on lung development have been well established, the role of type 2 responses in postnatal lung development remains poorly understood. Recent studies indicate that type 2-associated immune cells, such as group 2 innate lymphoid cells and alveolar macrophages, are increased in number during postnatal alveolarisation. Here, we present the current state of understanding of the postnatal stages of lung development and the key cell types and mediators known to be involved. We also provide an overview of how stem cells are involved in lung development and regeneration, and the negative influences of respiratory infections. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Emerging therapeutic potential of group 2 innate lymphoid cells in acute kidney injury.

Acute kidney injury (AKI) remains a global challenge and, despite the availability of dialysis and transplantation, can be fatal. Those that survive an AKI are at increased risk of developing chronic kidney disease and end stage renal failure. Understanding the fundamental mechanisms underpinning the pathophysiology of AKI is critical for developing novel strategies for diagnosis and treatment. A growing body of evidence indicates that amplifying type 2 immunity may have therapeutic potential in kidney injury and disease. Of particular interest are the recently described subset of innate immune cells, termed group 2 innate lymphoid cells (ILCs). Group 2 ILCs are crucial tissue-resident immune cells that maintain homeostasis and regulate tissue repair at multiple organ sites, including the kidney. They are critical mediators of type 2 immune responses following infection and injury. The existing literature suggests that activation of group 2 ILCs and production of a local type 2 immune milieu is protective against renal injury and associated pathology. In this review, we describe the emerging role for group 2 ILCs in renal homeostasis and repair. We provide an in-depth discussion of the most recent literature that use preclinical models of AKI and assess the therapeutic effect of modulating group 2 ILC function. We debate the potential for targeting these cells as novel cellular therapies in AKI and discuss the implications for future studies and translation. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

IL-22 and its receptors are increased in human and experimental COPD and contribute to pathogenesis.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death globally. The lack of effective treatments results from an incomplete understanding of the underlying mechanisms driving COPD pathogenesis.Interleukin (IL)-22 has been implicated in airway inflammation and is increased in COPD patients. However, its roles in the pathogenesis of COPD is poorly understood. Here, we investigated the role of IL-22 in human COPD and in cigarette smoke (CS)-induced experimental COPD.IL-22 and IL-22 receptor mRNA expression and protein levels were increased in COPD patients compared to healthy smoking or non-smoking controls. IL-22 and IL-22 receptor levels were increased in the lungs of mice with experimental COPD compared to controls and the cellular source of IL-22 included CD4+ T-helper cells, γδ T-cells, natural killer T-cells and group 3 innate lymphoid cells. CS-induced pulmonary neutrophils were reduced in IL-22-deficient (Il22 -/-) mice. CS-induced airway remodelling and emphysema-like alveolar enlargement did not occur in Il22 -/- mice. Il22 -/- mice had improved lung function in terms of airway resistance, total lung capacity, inspiratory capacity, forced vital capacity and compliance.These data highlight important roles for IL-22 and its receptors in human COPD and CS-induced experimental COPD.

TRAIL signals through the ubiquitin ligase MID1 to promote pulmonary fibrosis.

BACKGROUND: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has previously been demonstrated to play a pro-inflammatory role in allergic airways disease and COPD through the upregulation of the E3 ubiquitin ligase MID1 and the subsequent deactivation of protein phosphatase 2A (PP2A). METHODS: Biopsies were taken from eight IPF patients presenting to the Second Affiliated Hospital of Jilin University, China between January 2013 and February 2014 with control samples obtained from resected lung cancers. Serum TRAIL, MID1 protein and PP2A activity in biopsies, and patients' lung function were measured. Wild type and TRAIL deficient Tnfsf10-/- BALB/c mice were administered bleomycin to induce fibrosis and some groups were treated with the FTY720 analogue AAL(s) to activate PP2A. Mouse fibroblasts were treated with recombinant TRAIL and fibrotic responses were assessed. RESULTS: TRAIL in serum and MID1 protein levels in biopsies from IPF patients were increased compared to controls. MID1 levels were inversely associated while PP2A activity levels correlated with DLco. Tnfsf10-/- and mice treated with the PP2A activator AAL(s) were largely protected against bleomycin-induced reductions in lung function and fibrotic changes. Addition of recombinant TRAIL to mouse fibroblasts in-vitro increased collagen production which was reversed by PP2A activation with AAL(s). CONCLUSION: TRAIL signalling through MID1 deactivates PP2A and promotes fibrosis with corresponding lung function decline. This may provide novel therapeutic targets for IPF.

RelB-Deficient Dendritic Cells Promote the Development of Spontaneous Allergic Airway Inflammation.

RelB is a member of the NF-κB family, which is essential for dendritic cell (DC) function and maturation. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Here, we identify a pivotal role for RelB in the development of spontaneous AAI that is independent of exogenous allergen exposure. We assessed AAI in two strains of RelB-deficient (RelB-/-) mice: one with a targeted deletion and one expressing a major histocompatibility complex transgene. To determine the importance of RelB in DCs, RelB-sufficient DCs (RelB+/+ or RelB-/-) were adoptively transferred into RelB-/- mice. Both strains had increased pulmonary inflammation compared with their respective wild-type (RelB+/+) and heterozygous (RelB+/-) controls. RelB-/- mice also had increased inflammatory cell influx into the airways, levels of chemokines (CCL2/3/4/5/11/17 and CXCL9/10/13) and T-helper cell type 2-associated cytokines (IL-4/5) in lung tissues, serum IgE, and airway remodeling (mucus-secreting cell numbers, collagen deposition, and epithelial thickening). Transfer of RelB+/- CD11c+ DCs into RelB-/- mice decreased pulmonary inflammation, with reductions in lung chemokines, T-helper cell type 2-associated cytokines (IL-4/5/13/25/33 and thymic stromal lymphopoietin), serum IgE, type 2 innate lymphoid cells, myeloid DCs, γδ T cells, lung Vß13+ T cells, mucus-secreting cells, airway collagen deposition, and epithelial thickening. These data indicate that RelB deficiency may be a key pathway underlying AAI, and that DC-encoded RelB is sufficient to restore control of this inflammation.

Toll-like receptor 2 and 4 have opposing roles in the pathogenesis of cigarette smoke-induced chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death and imposes major socioeconomic burdens globally. It is a progressive and disabling condition that severely impairs breathing and lung function. There is a lack of effective treatments for COPD, which is a direct consequence of the poor understanding of the underlying mechanisms involved in driving the pathogenesis of the disease. Toll-like receptor (TLR)2 and TLR4 are implicated in chronic respiratory diseases, including COPD, asthma and pulmonary fibrosis. However, their roles in the pathogenesis of COPD are controversial and conflicting evidence exists. In the current study, we investigated the role of TLR2 and TLR4 using a model of cigarette smoke (CS)-induced experimental COPD that recapitulates the hallmark features of human disease. TLR2, TLR4, and associated coreceptor mRNA expression was increased in the airways in both experimental and human COPD. Compared with wild-type (WT) mice, CS-induced pulmonary inflammation was unaltered in TLR2-deficient ( Tlr2-/-) and TLR4-deficient ( Tlr4-/-) mice. CS-induced airway fibrosis, characterized by increased collagen deposition around small airways, was not altered in Tlr2-/- mice but was attenuated in Tlr4-/- mice compared with CS-exposed WT controls. However, Tlr2-/- mice had increased CS-induced emphysema-like alveolar enlargement, apoptosis, and impaired lung function, while these features were reduced in Tlr4-/- mice compared with CS-exposed WT controls. Taken together, these data highlight the complex roles of TLRs in the pathogenesis of COPD and suggest that activation of TLR2 and/or inhibition of TLR4 may be novel therapeutic strategies for the treatment of COPD.

Role for NLRP3 Inflammasome-mediated, IL-1ß-Dependent Responses in Severe, Steroid-Resistant Asthma.

RATIONALE: Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1ß responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma. OBJECTIVES: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1ß in severe, steroid-resistant asthma. METHODS: We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1ß responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1ß responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1ß antibody. Roles for IL-1ß-induced neutrophilic inflammation were examined using IL-1ß and anti-Ly6G. MEASUREMENTS AND MAIN RESULTS: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1ß responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1ß expression. Treatment with anti-IL-1ß, Ac-YVAD-cho, and MCC950 suppressed IL-1ß responses and the important steroid-resistant features of disease in mice, whereas IL-1ß administration recapitulated these features. Neutrophil depletion suppressed IL-1ß-induced steroid-resistant airway hyperresponsiveness. CONCLUSIONS: NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.

MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying phosphoinositide 3-kinase-mediated suppression of histone deacetylase 2.

BACKGROUND: Severe steroid-insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid-insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid-insensitive patients with eosinophil-enriched inflammation have also been described. The mechanisms that underpin infection-induced, severe steroid-insensitive asthma can be elucidated by using mouse models of disease. OBJECTIVE: We sought to develop representative mouse models of severe, steroid-insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. METHODS: Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin-induced, severe, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. RESULTS: Infection induced increases in the levels of microRNA (miRNA)-21 (miR-21) expression in the lung during SSIAAD, whereas expression of the miR-21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3-kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR-21-specific antagomir (Ant-21) increased phosphatase and tensin homolog levels. Treatment with Ant-21, or the pan-PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. CONCLUSION: We identify a previously unrecognized role for an miR-21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR-21 as a novel therapeutic target for the treatment of this form of asthma.

TRAIL signaling is proinflammatory and proviral in a murine model of rhinovirus 1B infection.

the aim of this study is to elucidate the role of TRAIL during rhinovirus (RV) infection in vivo. Naïve wild-type and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-deficient (Tnfsf10-/-) BALB/c mice were infected intranasally with RV1B. In separate experiments, Tnfsf10-/- mice were sensitized and challenged via the airway route with house dust mite (HDM) to induce allergic airways disease and then challenged with RVIB or UV-RVIB. Airway hyperreactivity (AHR) was invasively assessed as total airways resistance in response to increasing methacholine challenge and inflammation was assessed in bronchoalveolar lavage fluid at multiple time points postinfection. Chemokines were quantified by ELISA of whole lung lysates and viral load was determined by quantitative RT-PCR and tissue culture infective dose (TCID50). Human airway epithelial cells (BEAS2B) were infected with RV1B and stimulated with recombinant TRAIL or neutralizing anti-TRAIL antibodies and viral titer assessed by TCID50 HDM-challenged Tnfsf10-/- mice were protected against RV-induced AHR and had suppressed cellular infiltration in the airways upon RV infection. Chemokine C-X-C-motif ligand 2 (CXCL2) production was suppressed in naïve Tnfsf10-/- mice infected with RV1B, with less RV1B detected 24 h postinfection. This was associated with reduced apoptotic cell death and a reduction of interferon (IFN)-λ2/3 but not IFN-α or IFN-ß. TRAIL stimulation increased, whereas anti-TRAIL antibodies reduced viral replication in RV1B-infected BEAS2B cells in vitro. In conclusion, TRAIL promotes RV-induced AHR, inflammation and RV1B replication, implicating this molecule and its downstream signaling pathways as a possible target for the amelioration of RV1B-induced allergic and nonallergic lung inflammation and AHR.

Programmed Death Ligand 1 Promotes Early-Life Chlamydia Respiratory Infection-Induced Severe Allergic Airway Disease.

Chlamydia infections are frequent causes of respiratory illness, particularly pneumonia in infants, and are linked to permanent reductions in lung function and the induction of asthma. However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. In the current study, we investigated the role of programmed death (PD)-1 and its ligands PD-L1 and PD-L2 in promoting early-life Chlamydia respiratory infection, and infection-induced airway hyperresponsiveness (AHR) and severe allergic airway disease in later life. Infection increased PD-1 and PD-L1, but not PD-L2, mRNA expression in the lung. Flow cytometric analysis of whole lung homogenates identified monocytes, dendritic cells, CD4(+), and CD8(+) T cells as major sources of PD-1 and PD-L1. Inhibition of PD-1 and PD-L1, but not PD-L2, during infection ablated infection-induced AHR in later life. Given that PD-L1 was the most highly up-regulated and its targeting prevented infection-induced AHR, subsequent analyses focused on this ligand. Inhibition of PD-L1 had no effect on Chlamydia load but suppressed infection-induced pulmonary inflammation. Infection decreased the levels of the IL-13 decoy receptor in the lung, which were restored to baseline levels by inhibition of PD-L1. Finally, inhibition of PD-L1 during infection prevented subsequent infection-induced severe allergic airways disease in later life by decreasing IL-13 levels, Gob-5 expression, mucus production, and AHR. Thus, early-life Chlamydia respiratory infection-induced PD-L1 promotes severe inflammation during infection, permanent reductions in lung function, and the development of more severe allergic airway disease in later life.

TRAIL deficiency and PP2A activation with salmeterol ameliorates egg allergen-driven eosinophilic esophagitis.

Food antigens are common inflammatory triggers in pediatric eosinophilic esophagitis (EoE). TNF-related apoptosis-inducing ligand (TRAIL) promotes eosinophilic inflammation through the upregulation of the E3 ubiquitin ligase Midline (MID)-1 and subsequent downregulation of protein phosphatase 2A (PP2A), but the role of this pathway in EoE that is experimentally induced by repeated food antigen challenges has not been investigated. Esophageal mucosal biopsies were collected from children with EoE and controls and assessed for TRAIL and MID-1 protein and mRNA transcript levels. Wild-type and TRAIL-deficient (Tnfsf10-/-) mice were administered subcutaneous ovalbumin (OVA) followed by oral OVA challenges. In separate experiments, OVA-challenged mice were intraperitoneally administered salmeterol or dexamethasone. Esophageal biopsies from children with EoE revealed increased levels of TRAIL and MID-1 and reduced PP2A activation compared with controls. Tnfsf10-/- mice were largely protected from esophageal fibrosis, eosinophilic inflammation, and the upregulation of TSLP, IL-5, IL-13, and CCL11 when compared with wild-type mice. Salmeterol administration to wild-type mice with experimental EoE restored PP2A activity and also prevented esophageal eosinophilia, inflammatory cytokine expression, and remodeling, which was comparable to the treatment effect of dexamethasone. TRAIL and PP2A regulate inflammation and fibrosis in experimental EoE, which can be therapeutically modulated by salmeterol.