Hajera Amatullah

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

BackgroundArginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.MethodsTo investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3), or HEPA-filtered air (FA), for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.ResultsCompared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models.ConclusionsThis study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.

Comparative cardiopulmonary effects of size-fractionated airborne particulate matter

Context: Strong epidemiological evidence exists linking particulate matter (PM) exposures with hospital admissions of individuals for cardiopulmonary symptoms. The PM size is important in influencing the extent of infiltration into the respiratory tract and systemic circulation and directs the differential physiological impacts. Objective: To investigate the differential effects of the quasi-ultrafine (PM0.2), fine (PM0.15-2.5), and coarse PM (PM2.5-10) size fractions on pulmonary and cardiac function. Methods: Female BALB/c mice were exposed to HEPA-filtered laboratory air or concentrated coarse, fine, or quasi-ultrafine PM using Harvard Ambient Particle Concentrators in conjunction with our nose-only exposure system. These exposures were conducted as part of the “Health Effects of Aerosols in Toronto (HEAT)” campaign. Following a 4 h exposure, mice underwent assessment of respiratory function and recording of electrocardiograms using the flexiVent® system. Results: Exposure to coarse and fine PM resulted in a significant reduction in quasistatic compliance of the lung. Baseline total respiratory resistance and maximum responsiveness to methacholine were augmented after coarse PM exposures but were not affected by quasi-ultrafine PM exposures. In contrast, quasi-ultrafine PM alone had a significant effect on heart rate and in reducing heart rate variability. Conclusion: These findings indicate that coarse and fine PM influence lung function and airways responsiveness, while ultrafine PM can perturb cardiac function. This study supports the hypothesis that coarse and fine PM exerts its predominant physiologic effects at the site of deposition in the airways, whereas ultrafine PM likely crosses the alveolar epithelial barrier into the systemic circulation to affect cardiovascular function.

Spleen tyrosine kinase inhibition attenuates airway hyperresponsiveness and pollution-induced enhanced airway response in a chronic mouse model of asthma

BACKGROUND Asthma is a chronic inflammatory disease characterized by airways hyperresponsiveness (AHR), reversible airflow obstruction, airway remodeling, and episodic exacerbations caused by air pollutants, such as particulate matter (PM; PM <2.5 μm in diameter [PM(2.5)]) and ozone (O(3)). Spleen tyrosine kinase (Syk), an immunoregulatory kinase, has been implicated in the pathogenesis of asthma. OBJECTIVE We sought to evaluate the effect of Syk inhibition on AHR in a chronic mouse model of allergic airways inflammation and pollutant exposure. METHODS We used a 12-week chronic ovalbumin (OVA) sensitization and challenge mouse model of airways inflammation followed by exposure to PM(2.5) plus O(3). Respiratory mechanics and methacholine (MCh) responsiveness were assessed by using the flexiVent system. The Syk inhibitor NVP-QAB-205 was nebulized intratracheally by using a treatment-based protocol 15 minutes before assessment of MCh responsiveness. RESULTS Syk expression increased significantly in the airway epithelia of OVA-sensitized and OVA-challenged (OVA/OVA) mice compared with OVA-sensitized but PBS-challenged (OVA/PBS) control mice. OVA/OVA mice exhibited AHR to MCh, which was attenuated by a single administration of NVP-QAB-205 (0.3 and 3 mg/kg). PM(2.5) plus O(3) significantly augmented AHR to MCh in the OVA/OVA mice, which was abrogated by NVP-QAB-205. Total inflammatory cell counts were significantly higher in the bronchoalveolar lavage fluid from OVA/OVA than OVA/PBS mice and were unaffected by PM(2.5) plus O(3) or NVP-QAB-205. CONCLUSION NVP-QAB-205 reduced AHR and the enhanced response to PM(2.5) plus O(3) to normal levels in an established model of chronic allergic airways inflammation, suggesting that Syk inhibitors have promise as a therapy for asthma.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

ATF3 Protects Pulmonary Resident Cells from Acute and Ventilator-Induced Lung Injury by Preventing Nrf2 Degradation

AIMS Ventilator-induced lung injury (VILI) contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). Absence of activating transcription factor 3 (ATF3) confers susceptibility to ALI/VILI. To identify cell-specific ATF3-dependent mechanisms of susceptibility to ALI/VILI, we generated ATF3 chimera by adoptive bone marrow (BM) transfer and randomized to inhaled saline or lipopolysacharide (LPS) in the presence of mechanical ventilation (MV). Adenovirus vectors to silence or overexpress ATF3 were used in primary human bronchial epithelial cells and murine BM-derived macrophages from wild-type or ATF3-deficient mice. RESULTS Absence of ATF3 in myeloid-derived cells caused increased pulmonary cellular infiltration. In contrast, absence of ATF3 in parenchymal cells resulted in loss of alveolar-capillary membrane integrity and increased exudative edema. ATF3-deficient macrophages were unable to limit the expression of pro-inflammatory mediators. Knockdown of ATF3 in resident cells resulted in decreased junctional protein expression and increased paracellular leak. ATF3 overexpression abrogated LPS induced membrane permeability. Despite release of ATF3-dependent Nrf2 transcriptional inhibition, mice that lacked ATF3 expression in resident cells had increased Nrf2 protein degradation. INNOVATION In our model, in the absence of ATF3 in parenchymal cells increased Nrf2 degradation is the result of increased Keap-1 expression and loss of DJ-1 (Parkinson disease [autosomal recessive, early onset] 7), previously not known to play a role in lung injury. CONCLUSION Results suggest that ATF3 confers protection to lung injury by preventing inflammatory cell recruitment and barrier disruption in a cell-specific manner, opening novel opportunities for cell specific therapy for ALI/VILI.

DJ-1/PARK7 Impairs Bacterial Clearance in Sepsis

Rationale: Effective and rapid bacterial clearance is a fundamental determinant of outcomes in sepsis. DJ‐1 is a well‐established reactive oxygen species (ROS) scavenger. Objectives: Because cellular ROS status is pivotal to inflammation and bacterial killing, we determined the role of DJ‐1 in bacterial sepsis. Methods: We used cell and murine models with gain‐ and loss‐of‐function experiments, plasma, and cells from patients with sepsis. Measurements and Main Results: Stimulation of bone marrow‐derived macrophages (BMMs) with endotoxin resulted in increased DJ‐1 mRNA and protein expression. Cellular and mitochondrial ROS was increased in DJ‐1‐deficient (−/−) BMMs compared with wild‐type. In a clinically relevant model of polymicrobial sepsis (cecal ligation and puncture), DJ‐1−/− mice had improved survival and bacterial clearance. DJ‐1−/− macrophages exhibited enhanced phagocytosis and bactericidal activity in vitro, and adoptive transfer of DJ‐1−/− bone marrow‐derived mononuclear cells rescued wild‐type mice from cecal ligation and puncture‐induced mortality. In stimulated BMMs, DJ‐1 inhibited ROS production by binding to p47phox, a critical component of the NADPH oxidase complex, disrupting the complex and facilitating Nox2 (gp91phox) ubiquitination and degradation. Knocking down DJ‐1 (siRNA) in THP‐1 (human monocytic cell line) and polymorphonuclear cells from patients with sepsis enhanced bacterial killing and respiratory burst. DJ‐1 protein levels were elevated in plasma from patients with sepsis. Higher levels of circulating DJ‐1 were associated with increased organ failure and death. Conclusions: These novel findings reveal DJ‐1 impairs optimal ROS production for bacterial killing with important implications for host survival in sepsis.

Maintenance of macrophage transcriptional programs and intestinal homeostasis by epigenetic reader SP140

The epigenetic reader SP140, which is polymorphic in Crohn’s disease patients, regulates transcriptional programs in macrophages. PHDs crack the histone code Epigenetic readers that recognize histone modifications facilitate histone code–based transcriptional programming. Bromodomain- and plant homeodomain (PHD)–containing proteins often serve as readers of acetylation or methylation on histones, respectively. Mehta et al. have examined the function of SP140, a bromodomain- and PHD domain–containing reader in immune cells, and report that SP140 plays an essential role in repressing expression of lineage-inappropriate genes in macrophages. The authors also propose that SP140 polymorphisms associated with the development of Crohn’s disease represent functional loss of SP140. These studies advance our understanding of how epigenetic readers regulate immune responses in normal and diseased states. Epigenetic “readers” that recognize defined posttranslational modifications on histones have become desirable therapeutic targets for cancer and inflammation. SP140 is one such bromodomain- and plant homeodomain (PHD)–containing reader with immune-restricted expression, and single-nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unclear. We show that SP140 is critical for transcriptional programs that uphold the macrophage state. SP140 preferentially occupies promoters of silenced, lineage-inappropriate genes bearing the histone modification H3K27me3, such as the HOXA cluster in human macrophages, and ensures their repression. Depletion of SP140 in mouse or human macrophages resulted in severely compromised microbe-induced activation. We reveal that peripheral blood mononuclear cells (PBMCs) or B cells from individuals carrying CD-associated SNPs within SP140 have defective SP140 messenger RNA splicing and diminished SP140 protein levels. Moreover, CD patients carrying SP140 SNPs displayed suppressed innate immune gene signatures in a mixed population of PBMCs that stratified them from other CD patients. Hematopoietic-specific knockdown of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)–induced colitis, and low SP140 levels in human CD intestinal biopsies correlated with relatively lower intestinal innate cytokine levels and improved response to anti–tumor necrosis factor (TNF) therapy. Thus, the epigenetic reader SP140 is a key regulator of macrophage transcriptional programs for cellular state, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by ineffective innate immunity, normally critical for intestinal homeostasis.

Examining the role of arginase in air pollution-induced exacerbation of asthma

Background The arginase isozymes (arginase 1 and 2), convert Larginine into L-ornithine and urea, and thus compete with the nitric oxide synthase isozymes for substrate. We have previously shown that arginase 1 expression is upregulated in human asthma and plays a functional role in airways hyperresponsiveness (AHR) in an animal model of allergic airways inflammation. Ambient particles and ozone are major constituents of urban air pollution and contribute to asthma exacerbations. However, the mechanisms underlying the exacerbation of allergic airways disease by air pollution remain to be elucidated. There is evidence that arginase expression is augmented in cigarette smoking asthmatics. We tested the hypothesis that arginase is involved in the exacerbation of respiratory symptoms in response to air pollution in animal models of allergic airways inflammation.