Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b+ macrophage subpopulations and aggravates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.

Genomic classification and antimicrobial resistance profiling of Streptococcus pneumoniae and Haemophilus influenza isolates associated with paediatric otitis media and upper respiratory infection.

Acute otitis media (AOM) is the most common childhood bacterial infectious disease requiring antimicrobial therapy. Most cases of AOM are caused by translocation of Streptococcus pneumoniae or Haemophilus influenzae from the nasopharynx to the middle ear during an upper respiratory tract infection (URI). Ongoing genomic surveillance of these pathogens is important for vaccine design and tracking of emerging variants, as well as for monitoring patterns of antibiotic resistance to inform treatment strategies and stewardship.In this work, we examined the ability of a genomics-based workflow to determine microbiological and clinically relevant information from cultured bacterial isolates obtained from patients with AOM or an URI. We performed whole genome sequencing (WGS) and analysis of 148 bacterial isolates cultured from the nasopharynx (N = 124, 94 AOM and 30 URI) and ear (N = 24, all AOM) of 101 children aged 6-35 months presenting with AOM or an URI. We then performed WGS-based sequence typing and antimicrobial resistance profiling of each strain and compared results to those obtained from traditional microbiological phenotyping.WGS of clinical isolates resulted in 71 S. pneumoniae genomes and 76 H. influenzae genomes. Multilocus sequencing typing (MSLT) identified 33 sequence types for S. pneumoniae and 19 predicted serotypes including the most frequent serotypes 35B and 3. Genome analysis predicted 30% of S. pneumoniae isolates to have complete or intermediate penicillin resistance. AMR predictions for S. pneumoniae isolates had strong agreement with clinical susceptibility testing results for beta-lactam and non beta-lactam antibiotics, with a mean sensitivity of 93% (86-100%) and a mean specificity of 98% (94-100%). MLST identified 29 H. influenzae sequence types. Genome analysis identified beta-lactamase genes in 30% of H. influenzae strains, which was 100% in agreement with clinical beta-lactamase testing. We also identified a divergent highly antibiotic-resistant strain of S. pneumoniae, and found its closest sequenced strains, also isolated from nasopharyngeal samples from over 15 years ago.Ultimately, our work provides the groundwork for clinical WGS-based workflows to aid in detection and analysis of H. influenzae and S. pneumoniae isolates.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

Potentiation of long-acting ß2-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP.

INTRODUCTION: Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HYPOTHESIS: Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. METHODS: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. RESULTS: Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. CONCLUSION: Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

IL-4Rα blockade reduces influenza-associated morbidity in a murine model of allergic asthma.

BACKGROUND: Asthma was identified as the most common comorbidity in hospitalized patients during the 2009 H1N1 influenza pandemic. We determined using a murine model of allergic asthma whether these mice experienced increased morbidity from pandemic H1N1 (pH1N1) viral infection and whether blockade of interleukin-4 receptor α (IL-4Rα), a critical mediator of Th2 signalling, improved their outcomes. METHODS: Male BALB/c mice were intranasally sensitized with house dust mite antigen (Der p 1) for 2 weeks; the mice were then inoculated intranasally with a single dose of pandemic H1N1 (pH1N1). The mice were administered intraperitoneally anti-IL-4Rα through either a prophylactic or a therapeutic treatment strategy. RESULTS: Infection with pH1N1 of mice sensitized to house dust mite (HDM) led to a 24% loss in weight by day 7 of infection (versus 14% in non-sensitized mice; p < .05). This was accompanied by increased viral load in the airways and a dampened anti-viral host responses to the infection. Treatment of HDM sensitized mice with a monoclonal antibody against IL-4Rα prior to or following pH1N1 infection prevented the excess weight loss, reduced the viral load in the lungs and ameliorated airway eosinophilia and systemic inflammation related to the pH1N1 infection. CONCLUSION: Together, these data implicate allergic asthma as a significant risk factor for H1N1-related morbidity and reveal a potential therapeutic role for IL-4Rα signalling blockade in reducing the severity of influenza infection in those with allergic airway disease.

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.

Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2.

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.

Predicting the recombination potential of severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged to cause widespread infections in humans. SARS-CoV-2 infections have been reported in the Kingdom of Saudi Arabia, where Middle East respiratory syndrome coronavirus (MERS-CoV) causes seasonal outbreaks with a case fatality rate of ~37 %. Here we show that there exists a theoretical possibility of future recombination events between SARS-CoV-2 and MERS-CoV RNA. Through computational analyses, we have identified homologous genomic regions within the ORF1ab and S genes that could facilitate recombination, and have analysed co-expression patterns of the cellular receptors for SARS-CoV-2 and MERS-CoV, ACE2 and DPP4, respectively, to identify human anatomical sites that could facilitate co-infection. Furthermore, we have investigated the likely susceptibility of various animal species to MERS-CoV and SARS-CoV-2 infection by comparing known virus spike protein-receptor interacting residues. In conclusion, we suggest that a recombination between SARS-CoV-2 and MERS-CoV RNA is possible and urge public health laboratories in high-risk areas to develop diagnostic capability for the detection of recombined coronaviruses in patient samples.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

NLRP3 deficiency exacerbates enterovirus infection in mice.

The role for the NOD-like receptor (NLR) P3 inflammasome in enterovirus infection remains controversial. Available data suggest that the NLRP3 inflammasome is protective against enterovirus A71 but detrimental to the host during coxsackievirus B3 (CVB3) infection. CVB3 is a common etiologic agent associated with myocarditis and pancreatitis. Previous findings on the role of NLRP3 in CVB3 were based primarily on indirect evidence. Here, we utilized NLRP3 knockout mice as well as immune and cardiac cells to investigate the direct interplay between CVB3 infection and NLRP3 activation. We demonstrated that NLRP3 knockout mice exhibited more severe disease phenotype after CVB3 infection (significantly higher virus titers), increased myocardial, and pancreatic damage, as well as markedly impaired cardiac function compared to nontransgenic control mice. We further showed that NLRP3 activity was enhanced during early stage of CVB3 infection, as evidenced by increased gene expression and/or secretion of IL-1ß and caspase-1. Finally, we demonstrated that CVB3 inactivates the NLRP3 inflammasome by degrading NLRP3 and its upstream serine/threonine-protein kinase receptor-interacting protein 1/3 via the proteolytic activity of virus-encoded proteinases. Taken together, our results reveal the functional significance of NLRP3 in host antiviral immunity against CVB3 infection and the mechanisms by which CVB3 has evolved to counteract the host defense response.-Wang, C., Fung, G., Deng, H., Jagdeo, J., Mohamud, Y., Xue, Y. C., Jan, E., Hirota, J. A., Luo, H. NLRP3 deficiency exacerbates enterovirus infection in mice.

An update on immunologic mechanisms in the respiratory mucosa in response to air pollutants.

Every day, we breathe in more than 10,000 L of air that contains a variety of air pollutants that can pose negative consequences to lung health. The respiratory mucosa formed by the airway epithelium is the first point of contact for air pollution in the lung, functioning as a mechanical and immunologic barrier. Under normal circumstances, airway epithelial cells connected by tight junctions secrete mucus, airway surface lining fluid, host defense peptides, and antioxidants and express innate immune pattern recognition receptors to respond to inhaled foreign substances and pathogens. Under conditions of air pollution exposure, the defenses of the airway epithelium are compromised by reductions in barrier function, impaired host defense to pathogens, and exaggerated inflammatory responses. Central to the mechanical and immunologic changes induced by air pollution are activation of redox-sensitive pathways and a role for antioxidants in normalizing these negative effects. Genetic variants in genes important in epithelial cell function and phenotype contribute to a diversity of responses to air pollution in the population at the individual and group levels and suggest a need for personalized approaches to attenuate the respiratory mucosal immune responses to air pollution.

Inhibition of Marfan Syndrome Aortic Root Dilation by Losartan: Role of Angiotensin II Receptor Type 1-Independent Activation of Endothelial Function.

Marfan syndrome (MFS) is a genetic disorder that frequently leads to aortic root dissection and aneurysm. Despite promising preclinical and pilot clinical data, a recent large-scale study using antihypertensive angiotensin II (AngII) receptor type 1 (ATR1) blocker losartan has failed to meet expectations at preventing MFS-associated aortic root dilation, casting doubts about optimal therapy. To study the deleterious role of normal ATR1 signaling in aortic root widening, we generated MFS mice lacking ATR1a expression in an attempt to preserve protective ATR2 signaling. Despite being hypotensive and resistant to AngII vasopressor effects, MFS/ATR1a-null mice showed unabated aortic root enlargement and remained fully responsive to losartan, confirming that blood pressure lowering is of minor therapeutic value in MFS and that losartan's antiremodeling properties may be ATR1 independent. Having shown that MFS causes endothelial dysfunction and that losartan can activate endothelial function in mice and patients, we found that nitric oxide synthase (NOS) inhibition renders losartan therapeutically inactive, whereas multiple transgenic and pharmacologic models of endothelial NOS activation block aortic root dilation by correcting extracellular signal-regulated kinase signaling. In vitro, losartan can increase endothelial NO release in the absence of AngII and correct MFS NO levels in vivo. Our data suggest that increased protective endothelial function, rather than ATR1 inhibition or blood pressure lowering, might be of therapeutic significance in preventing aortic root disease in MFS.

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.

Attenuating immune pathology using a microbial-based intervention in a mouse model of cigarette smoke-induced lung inflammation.

BACKGROUND: Cigarette smoke exposure is the major risk factor for developing COPD. Presently, available COPD treatments focus on suppressing inflammation and providing bronchodilation. However, these options have varying efficacy in controlling symptoms and do not reverse or limit the progression of COPD. Treatments strategies using bacterial-derived products have shown promise in diseases characterized by inflammation and immune dysfunction. This study investigated for the first time whether a novel immunotherapy produced from inactivated Klebsiella (hereafter referred to as KB) containing all the major Klebsiella macromolecules, could attenuate cigarette smoke exposure-induced immune responses. We hypothesized that KB, by re-directing damaging immune responses, would attenuate cigarette smoke-induced lung inflammation and bronchoalveolar (BAL) cytokine and chemokine production. METHODS: KB was administered via a subcutaneous injection prophylactically before initiating a 3-week acute nose-only cigarette smoke exposure protocol. Control mice received placebo injection and room air. Total BAL and differential cell numbers were enumerated. BAL and serum were analysed for 31 cytokines, chemokines, and growth factors. Lung tissue and blood were analysed for Ly6CHI monocytes/macrophages and neutrophils. Body weight and clinical scores were recorded throughout the experiment. RESULTS: We demonstrate that KB treatment attenuated cigarette smoke-induced lung inflammation as shown by reductions in levels of BAL IFNγ, CXCL9, CXCL10, CCL5, IL-6, G-CSF, and IL-17. KB additionally attenuated the quantity of BAL lymphocytes and macrophages. In parallel to the attenuation of lung inflammation, KB induced a systemic immune activation with increases in Ly6CHI monocytes/macrophages and neutrophils. CONCLUSIONS: This is the first demonstration that subcutaneous administration of a microbial-based immunotherapy can attenuate cigarette smoke-induced lung inflammation, and modulate BAL lymphocyte and macrophage levels, while inducing a systemic immune activation and mobilization. These data provide a foundation for future studies exploring how KB may be used to either reverse or prevent progression of established emphysema and small airways disease associated with chronic cigarette smoke exposure. The data suggest the intriguing possibility that KB, which stimulates rather than suppresses systemic immune responses, might be a novel means by which the course of COPD pathogenesis may be altered.

Animal models of COPD: What do they tell us?

COPD is a major cause of global mortality and morbidity but current treatments are poorly effective. This is because the underlying mechanisms that drive the development and progression of COPD are incompletely understood. Animal models of disease provide a valuable, ethically and economically viable experimental platform to examine these mechanisms and identify biomarkers that may be therapeutic targets that would facilitate the development of improved standard of care. Here, we review the different established animal models of COPD and the various aspects of disease pathophysiology that have been successfully recapitulated in these models including chronic lung inflammation, airway remodelling, emphysema and impaired lung function. Furthermore, some of the mechanistic features, and thus biomarkers and therapeutic targets of COPD identified in animal models are outlined. Some of the existing therapies that suppress some disease symptoms that were identified in animal models and are progressing towards therapeutic development have been outlined. Further studies of representative animal models of human COPD have the strong potential to identify new and effective therapeutic approaches for COPD.

Gene expression analysis in asthma using a targeted multiplex array.

BACKGROUND: Gene expression changes in the structural cells of the airways are thought to play a role in the development of asthma and airway hyperresponsiveness. This includes changes to smooth muscle contractile machinery and epithelial barrier integrity genes. We used a targeted gene expression arrays to identify changes in the expression and co-expression of genes important in asthma pathology. METHODS: RNA was isolated from the airways of donor lungs from 12 patients with asthma (8 fatal) and 12 non-asthmatics controls and analyzed using a multiplexed, hypothesis-directed platform to detect differences in gene expression. Genes were grouped according to their role in airway dysfunction: airway smooth muscle contraction, cytoskeleton structure and regulation, epithelial barrier function, innate and adaptive immunity, fibrosis and remodeling, and epigenetics. RESULTS: Differential gene expression and gene co-expression analyses were used to identify disease associated changes in the airways of asthmatics. There was significantly decreased abundance of integrin beta 6 and Ras-Related C3 Botulinum Toxin Substrate 1 (RAC1) in the airways of asthmatics, genes which are known to play an important role in barrier function. Significantly elevated levels of Collagen Type 1 Alpha 1 (COL1A1) and COL3A1 which have been shown to modulate cell proliferation and inflammation, were found in asthmatic airways. Additionally, we identified patterns of differentially co-expressed genes related to pathways involved in virus recognition and regulation of interferon production. 7 of 8 pairs of differentially co-expressed genes were found to contain CCCTC-binding factor (CTCF) motifs in their upstream promoters. CONCLUSIONS: Changes in the abundance of genes involved in cell-cell and cell-matrix interactions could play an important role in regulating inflammation and remodeling in asthma. Additionally, our results suggest that alterations to the binding site of the transcriptional regulator CTCF could drive changes in gene expression in asthmatic airways. Several asthma susceptibility loci are known to contain CTCF motifs and so understanding the role of this transcription factor may expand our understanding of asthma pathophysiology and therapeutic options.

Controlled diesel exhaust and allergen coexposure modulates microRNA and gene expression in humans: Effects on inflammatory lung markers.

BACKGROUND: Air pollution's association with asthma may be due to its augmentation of allergenic effects, but the role of microRNA (miRNA) and gene expression in this synergy is unknown. OBJECTIVE: We sought to determine whether exposure to allergen, exposure to diesel exhaust (DE), or coexposures modulate miRNA, gene expression, or inflammatory pathways and whether these measurements are correlated. METHODS: Fifteen participants with atopy completed this controlled study of 2 hours of filtered air or DE (300 µg PM2.5/m3) exposure, followed by saline-controlled segmental bronchial allergen challenge. Gene and miRNA expression in bronchial brushings and lung inflammatory markers were measured 48 hours later, in study arms separated by approximately 4 weeks. Expression of miRNAs, messenger RNAs, and inflammatory markers and their interrelationships were determined using regression. RESULTS: Robust linear models indicated that DE plus saline and DE plus allergen significantly modulated the highest number of miRNAs and messenger RNAs, respectively, relative to control (filtered air plus saline). In mixed models, allergen exposure modulated (q ≤ 0.2) miRNAs including miR-183-5p, miR-324-5p, and miR-132-3p and genes including NFKBIZ and CDKN1A, but DE did not significantly modify this allergenic effect. Repression of CDKN1A by allergen-induced miR-132-3p may contribute to shedding of bronchial epithelial cells. CONCLUSIONS: Expression of specific miRNAs and genes associated with bronchial immune responses were significantly modulated by DE or allergen. However, DE did not augment the effect of allergen at 48 hours, suggesting that adjuvancy may be transient or require higher or prolonged exposure. In silico analysis suggested a possible mechanism contributing to epithelial wall damage following allergen exposure.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BACKGROUND: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects. METHODS: Twelve atopic subjects were exposed to DE 300 µg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa. RESULTS: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031). CONCLUSIONS: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Effect of diesel exhaust inhalation on blood markers of inflammation and neurotoxicity: a controlled, blinded crossover study.

CONTEXT: Epidemiological studies and animal research have suggested that air pollution may negatively impact the central nervous system (CNS). Controlled human exposure studies of the effect of air pollution on the brain have potential to enhance our understanding of this relationship and to inform potential biological mechanisms. OBJECTIVES: Biomarkers of systemic and CNS inflammation may address whether air pollution exposure induces inflammation, with potential for CNS negative effects. MATERIALS AND METHODS: Twenty-seven healthy adults were exposed to two conditions: filtered air (FA) and diesel exhaust (DE) (300 µg PM2.5/m(3)) for 120 min, in a double-blinded crossover study with exposures separated by four weeks. Prior to and at 0, 3, and 24 h following each exposure, serum and plasma were collected and analyzed for inflammatory cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α), the astrocytic protein S100b, the neuronal cytoplasmic enzyme neuron-specific enolase (NSE), and serum brain-derived neurotrophic factor (BDNF). We hypothesized that IL-6, TNF-α, S100b and NSE would increase, and BDNF would decrease, following DE exposure. RESULTS: At no time-point following exposure to DE was a significant increase in concentration from baseline seen for IL-6, TNF-α, S100b, or NSE relative to FA exposure. Similarly, no significant decrease in BDNF concentration from baseline was seen following DE exposure, relative to FA. Furthermore, the repeated measures ANOVA considered for all time-points and biomarkers revealed no significant time-exposure interaction. DISCUSSION AND CONCLUSION: These results suggest that short-term exposure to DE amongst healthy adults does not acutely affect the systemic or CNS biomarkers that we measured.