Dr Alexandra Brown

Lung transcriptomics studies in asthma have provided valuable information in the whole lung context, however, deciphering the individual contributions of the airway and parenchyma in disease pathogenesis may expedite the development of novel targeted treatment strategies. In this study, we performed transcriptomics on the airway and parenchyma using a house dust mite (HDM)-induced model of experimental asthma that replicates key features of the human disease. HDM exposure increased the expression of 3,255 genes, of which 212 were uniquely increased in the airways, 856 uniquely increased in the parenchyma, and 2187 commonly increased in both compartments. Further interrogation of these genes using a combination of network and transcription factor enrichment analyses identified several transcription factors that regulate airway and/or parenchymal gene expression, including transcription factor EC (TFEC), transcription factor PU.1 (SPI1), H2.0-like homeobox (HLX), metal response element binding transcription factor-1 (MTF1) and E74-like factor 4 (ets domain transcription factor, ELF4) involved in controlling innate immune responses. We next assessed the effects of inhibiting lung SPI1 responses using commercially available DB1976 and DB2313 on key disease outcomes. We found that both compounds had no protective effects on airway inflammation, however DB2313 (8¿mg/kg) decreased mucus secreting cell number, and both DB2313 (1¿mg/kg) and DB1976 (2.5¿mg/kg and 1¿mg/kg) reduced small airway collagen deposition. Significantly, both compounds decreased airway hyperresponsiveness. This study demonstrates that SPI1 is important in HDM-induced experimental asthma and that its pharmacological inhibition reduces HDM-induced airway collagen deposition and hyperresponsiveness.

The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI. JAK1 activation was also inhibited by OI in response to IL-13, interferon-ß, and interferon-¿ in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation in vivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.

Background and aims: Observational studies have demonstrated that the pneumococcal polysaccharide vaccine (PPV) is associated with reduced risk of cardiovascular events. This may be mediated through IgM antibodies to OxLDL, which have previously been associated with cardioprotective effects. The Australian Study for the Prevention through Immunisation of Cardiovascular Events (AUSPICE) is a double-blind, randomised controlled trial (RCT) of PPV in preventing ischaemic events. Participants received PPV or placebo once at baseline and are being followed-up for incident fatal and non-fatal myocardial infarction or stroke over 6 years. Methods: A subgroup of participants at one centre (Canberra; n = 1,001) were evaluated at 1 month and 2 years post immunisation for changes in surrogate markers of atherosclerosis, as pre-specified secondary outcomes: high-sensitive C-reactive protein (CRP), pulse wave velocity (PWV), and carotid intima-media thickness (CIMT). In addition, 100 participants were randomly selected in each of the intervention and control groups for measurement of anti-pneumococcal antibodies (IgG, IgG2, IgM) as well as anti-OxLDL antibodies (IgG and IgM to CuOxLDL, MDA-LDL, and PC-KLH). Results: Concentrations of anti-pneumococcal IgG and IgG2 increased and remained high at 2 years in the PPV group compared to the placebo group, while IgM increased and then declined, but remained detectable, at 2 years. There were statistically significant increases in all anti-OxLDL IgM antibodies at 1 month, which were no longer detectable at 2 years; there was no increase in anti-OxLDL IgG antibodies. There were no significant changes in CRP, PWV or CIMT between the treatment groups at the 2-year follow-up. Conclusions: PPV engenders a long-lasting increase in anti-pneumococcal IgG, and to a lesser extent, IgM titres, as well as a transient increase in anti-OxLDL IgM antibodies. However, there were no detectable changes in surrogate markers of atherosclerosis at the 2-year follow-up. Long-term, prospective follow-up of clinical outcomes is continuing to assess if PPV reduces CVD events.

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.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains. IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

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.

Background and objective Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and inflammation. Airway bacterial colonization is increased in COPD; however, the role of potentially pathogenic and non-pathogenic bacteria in the pathogenesis of disease is unclear. This study characterized the presence of bacteria in a well-characterized cohort of adults with COPD and healthy controls. Methods Adults with COPD (n = 70) and healthy controls (n = 51) underwent clinical assessment and sputum induction. Sputum was dispersed, and total and differential cell counts were performed. Bacteria were cultured, identified and enumerated. Supernatants were assessed for neutrophil elastase (NE) and IL-1ß. Common respiratory pathogens were also determined using real-time PCR. Results Participants with COPD had a typical neutrophilic inflammatory profile. The total load of bacteria was increased in COPD and was associated with poorer respiratory health status, as measured by the St George's Respiratory Questionnaire (Spearman's r = 0.336, P = 0.013). Significantly lower levels of culturable Bacillus species were identified compared with healthy controls. PCR analyses revealed increased rates of detection of potentially pathogenic bacteria with Haemophilus influenzae detection associated with higher sputum levels of NE and IL-1ß, while Streptococcus pneumoniae was more common in male ex-smokers with emphysema and a deficit in diffusion capacity. Conclusion Non-pathogenic and pathogenic bacteria were altered in the sputum of patients with COPD. These observations highlight the potential to identify treatment and management strategies that both target specific bacterial pathogens and restore the microbial balance, which may lead to reductions in inflammation and subsequent improvements in lung health.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM 10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM 10 -induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.