Cross-talk between IL-6 trans-signaling and AIM2 inflammasome/IL-1ß axes bridge innate immunity and epithelial apoptosis to promote emphysema.

Pulmonary emphysema is associated with dysregulated innate immune responses that promote chronic pulmonary inflammation and alveolar apoptosis, culminating in lung destruction. However, the molecular regulators of innate immunity that promote emphysema are ill-defined. Here, we investigated whether innate immune inflammasome complexes, comprising the adaptor ASC, Caspase-1 and specific pattern recognition receptors (PRRs), promote the pathogenesis of emphysema. In the lungs of emphysematous patients, as well as spontaneous gp130F/F and cigarette smoke (CS)-induced mouse models of emphysema, the expression (messenger RNA and protein) and activation of ASC, Caspase-1, and the inflammasome-associated PRR and DNA sensor AIM2 were up-regulated. AIM2 up-regulation in emphysema coincided with the biased production of the mature downstream inflammasome effector cytokine IL-1ß but not IL-18. These observations were supported by the genetic blockade of ASC, AIM2, and the IL-1 receptor and therapy with AIM2 antagonistic suppressor oligonucleotides, which ameliorated emphysema in gp130F/F mice by preventing elevated alveolar cell apoptosis. The functional requirement for AIM2 in driving apoptosis in the lung epithelium was independent of its expression in hematopoietic-derived immune cells and the recruitment of infiltrating immune cells in the lung. Genetic and inhibitor-based blockade of AIM2 also protected CS-exposed mice from pulmonary alveolar cell apoptosis. Intriguingly, IL-6 trans-signaling via the soluble IL-6 receptor, facilitated by elevated levels of IL-6, acted upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema. Collectively, we reveal cross-talk between the AIM2 inflammasome/IL-1ß and IL-6 trans-signaling axes for potential exploitation as a therapeutic strategy for emphysema.

Dual inhibition of airway inflammation and fibrosis by common ß cytokine receptor blockade.

BACKGROUND: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common ß (ßc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. OBJECTIVE: To determine the pathogenesis of ßc receptor-mediated inflammation and remodeling in severe asthma and to investigate ßc antagonism as a therapeutic strategy for mixed granulocytic airway disease. METHODS: ßc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human ßc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of ßc was used to block ßc signaling. RESULTS: ßc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. CONCLUSIONS: ßc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-ßc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.

pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Influenza A virus elicits peri-vascular adipose tissue inflammation and vascular dysfunction of the aorta in pregnant mice.

Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1-3 days post infection (d.p.i) with the levels being ~4-8 fold higher compared with the vessel wall. IAV infection also increased Ly6Clow patrolling monocytes and Ly6Chigh pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a "vascular storm"- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4+ and CD8+ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.

Early-life house dust mite aeroallergen exposure augments cigarette smoke-induced myeloid inflammation and emphysema in mice.

BACKGROUND: Longitudinal studies have identified childhood asthma as a risk factor for obstructive pulmonary disease (COPD) and asthma-COPD overlap (ACO) where persistent airflow limitation can develop more aggressively. However, a causal link between childhood asthma and COPD/ACO remains to be established. Our study aimed to model the natural history of childhood asthma and COPD and to investigate the cellular/molecular mechanisms that drive disease progression. METHODS: Allergic airways disease was established in three-week-old young C57BL/6 mice using house dust mite (HDM) extract. Mice were subsequently exposed to cigarette smoke (CS) and HDM for 8 weeks. Airspace enlargement (emphysema) was measured by the mean linear intercept method. Flow cytometry was utilised to phenotype lung immune cells. Bulk RNA-sequencing was performed on lung tissue. Volatile organic compounds (VOCs) in bronchoalveolar lavage-fluid were analysed to screen for disease-specific biomarkers. RESULTS: Chronic CS exposure induced emphysema that was significantly augmented by HDM challenge. Increased emphysematous changes were associated with more abundant immune cell lung infiltration consisting of neutrophils, interstitial macrophages, eosinophils and lymphocytes. Transcriptomic analyses identified a gene signature where disease-specific changes induced by HDM or CS alone were conserved in the HDM-CS group, and further revealed an enrichment of Mmp12, Il33 and Il13, and gene expression consistent with greater expansion of alternatively activated macrophages. VOC analysis also identified four compounds increased by CS exposure that were paradoxically reduced in the HDM-CS group. CONCLUSIONS: Early-life allergic airways disease worsened emphysematous lung pathology in CS-exposed mice and markedly alters the lung transcriptome.

Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages.

mRNA lipid nanoparticle (LNP) technology presents enormous opportunities to prevent and treat various diseases. Here, we developed a novel series of LNPs containing ionizable amino-lipids showing a remarkable array of tunable and pH-sensitive lyotropic liquid crystalline mesophases including the inverse bicontinuous cubic and hexagonal phases characterized by high-throughput synchrotron radiation X-ray scattering. Furthermore, with an interest in developing mRNA therapeutics for lung macrophage targeting, we discovered that there is a strong correlation between the mesophase transition of the LNPs during acidification and the macrophage association/transfection efficiency of mRNAs. The slight molecular structural differences between the SM-102 and ALC-0315 ionizable lipids are linked to the LNP's ability to transform their internal structures from an amorphous state to the inverse micellar, hexagonal, and finally cubic structures during endosomal maturation. SM-102 LNPs showed exceptionally improved transfection efficiency due to their ability to form a cubic structure at a lower pH than the ALC-0315 analogues, which remained within the hexagonal structure, previously attributed to promoting endosomal escape of the ionizable LNPs. Overall, the new knowledge draws our attention to the important role of mesophase transition in endosomal escape, and the novel LNP libraries reported herein have broad prospects for advancing mRNA therapeutics.

Cigarette smoke-induced pulmonary impairment is associated with social recognition memory impairments and alterations in microglial profiles within the suprachiasmatic nucleus of the hypothalamus.

Chronic obstructive pulmonary disease (COPD) is a major, incurable respiratory condition that is primarily caused by cigarette smoking (CS). Neurocognitive disorders including cognitive dysfunction, anxiety and depression are highly prevalent in people with COPD. It is understood that increased lung inflammation and oxidative stress from CS exposure may 'spill over' into the systemic circulation to promote the onset of these extra-pulmonary comorbidities, and thus impacts the quality of life of people with COPD. The precise role of the 'spill-over' of inflammation and oxidative stress in the onset of COPD-related neurocognitive disorders are unclear. The present study investigated the impact of chronic CS exposure on anxiety-like behaviors and social recognition memory, with a particular focus on the role of the 'spill-over' of inflammation and oxidative stress from the lungs. Adult male BALB/c mice were exposed to either room air (sham) or CS (9 cigarettes per day, 5 days a week) for 24 weeks and were either daily co-administered with the NOX2 inhibitor, apocynin (5 mg/kg, in 0.01 % DMSO diluted in saline, i.p.) or vehicle (0.01 % DMSO in saline) one hour before the initial CS exposure of the day. After 23 weeks, mice underwent behavioral testing and physiological diurnal rhythms were assessed by monitoring diurnal regulation profiles. Lungs were collected and assessed for hallmark features of COPD. Consistent with its anti-inflammatory and oxidative stress properties, apocynin treatment partially lessened lung inflammation and lung function decline in CS mice. CS-exposed mice displayed marked anxiety-like behavior and impairments in social recognition memory compared to sham mice, which was prevented by apocynin treatment. Apocynin was unable to restore the decreased Bmal1-positive cells, key in cells in diurnal regulation, in the suprachiasmatic nucleus of the hypothalamus to that of sham levels. CS-exposed mice treated with apocynin was associated with a restoration of microglial area per cell and basal serum corticosterone. This data suggests that we were able to model the CS-induced social recognition memory impairments seen in humans with COPD. The preventative effects of apocynin on memory impairments may be via a microglial dependent mechanism.

Influenza A virus causes maternal and fetal pathology via innate and adaptive vascular inflammation in mice.

Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.

Chronic obstructive pulmonary disease and atherosclerosis: common mechanisms and novel therapeutics.

Chronic obstructive pulmonary disease (COPD) and atherosclerosis are chronic irreversible diseases, that share a number of common causative factors including cigarette smoking. Atherosclerosis drastically impairs blood flow and oxygen availability to tissues, leading to life-threatening outcomes including myocardial infarction (MI) and stroke. Patients with COPD are most likely to die as a result of a cardiovascular event, with 30% of all COPD-related deaths being attributed to cardiovascular disease (CVD). Both atherosclerosis and COPD involve significant local (i.e. lung, vasculature) and systemic inflammation and oxidative stress, of which current pharmacological treatments have limited efficacy, hence the urgency for the development of novel life-saving therapeutics. Currently these diseases must be treated individually, with no therapies available that can effectively reduce the likelihood of comorbid CVD other than cessation of cigarette smoking. In this review, the important mechanisms that drive atherosclerosis and CVD in people with COPD are explained and we propose that modulation of both the oxidative stress and the inflammatory burden will provide a novel therapeutic strategy to treat both the pulmonary and systemic manifestations related to these diseases.

Ebselen abolishes vascular dysfunction in influenza A virus-induced exacerbations of cigarette smoke-induced lung inflammation in mice.

People with chronic obstructive pulmonary disease (COPD) are susceptible to respiratory infections which exacerbate pulmonary and/or cardiovascular complications, increasing their likelihood of death. The mechanisms driving these complications remain unknown but increased oxidative stress has been implicated. Here we investigated whether influenza A virus (IAV) infection, following chronic cigarette smoke (CS) exposure, worsens vascular function and if so, whether the antioxidant ebselen alleviates this vascular dysfunction. Male BALB/c mice were exposed to either room air or CS for 8 weeks followed by inoculation with IAV (Mem71, 1 × 104.5 pfu). Mice were treated with ebselen (10 mg/kg) or vehicle (5% w/v CM-cellulose in water) daily. Mice were culled 3- and 10-days post-infection, and their lungs lavaged to assess inflammation. The thoracic aorta was excised to investigate endothelial and smooth muscle dilator responses, expression of key vasodilatory and oxidative stress modulators, infiltrating immune cells and vascular remodelling. CS increased lung inflammation and caused significant vascular endothelial dysfunction, which was worsened by IAV infection. CS-driven increases in vascular oxidative stress, aortic wall remodelling and suppression of endothelial nitric oxide synthase (eNOS) were not affected by IAV infection. CS and IAV infection significantly enhanced T cell recruitment into the aortic wall. Ebselen abolished the exaggerated lung inflammation, vascular dysfunction and increased T cell infiltration in CS and IAV-infected mice. Our findings showed that ebselen treatment abolished vascular dysfunction in IAV-induced exacerbations of CS-induced lung inflammation indicating it may have potential for the treatment of cardiovascular comorbidities seen in acute exacerbations of COPD (AECOPD).

Targeting the human ßc receptor inhibits inflammatory myeloid cells and lung injury caused by acute cigarette smoke exposure.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a devastating disease commonly caused by cigarette smoke (CS) exposure that drives tissue injury by persistently recruiting myeloid cells into the lungs. A significant portion of COPD patients also present with overlapping asthma pathology including eosinophilic inflammation. The ßc cytokine family includes granulocyte monocyte-colony-stimulating factor, IL-5 and IL-3 that signal through their common receptor subunit ßc to promote the expansion and survival of multiple myeloid cells including monocytes/macrophages, neutrophils and eosinophils. METHODS: We have used our unique human ßc receptor transgenic (hßc Tg) mouse strain that expresses human ßc instead of mouse ßc and ßIL3 in an acute CS exposure model. Lung tissue injury was assessed by histology and measurement of albumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid. Transgenic mice were treated with an antibody (CSL311) that inhibits human ßc signalling. RESULTS: hßc Tg mice responded to acute CS exposure by expanding blood myeloid cell numbers and recruiting monocyte-derived macrophages (cluster of differentiation 11b+ [CD11b+ ] interstitial and exudative macrophages [IM and ExM]), neutrophils and eosinophils into the lungs. This inflammatory response was associated with lung tissue injury and oedema. Importantly, CSL311 treatment in CS-exposed mice markedly reduced myeloid cell numbers in the blood and BAL compartment. Furthermore, CSL311 significantly reduced lung CD11b+ IM and ExM, neutrophils and eosinophils, and this decline was associated with a significant reduction in matrix metalloproteinase-12 (MMP-12) and IL-17A expression, tissue injury and oedema. CONCLUSION: This study identifies CSL311 as a therapeutic antibody that potently inhibits immunopathology and lung injury caused by acute CS exposure.

Excess iron promotes emergence of foamy macrophages that overexpress ferritin in the lungs of silicosis patients.

BACKGROUND AND OBJECTIVE: Inhalation of high concentrations of respirable crystalline silica (RCS) can lead to silicosis. RCS contains varying levels of iron, which can cause oxidative stress and stimulate ferritin production. This study evaluated iron-related and inflammatory markers in control and silicosis patients. METHODS: A cohort of stone benchtop industry workers (n = 18) were radiologically classified by disease severity into simple or complicated silicosis. Peripheral blood and bronchoalveolar lavage (BAL) were collected to measure iron, ferritin, C-reactive protein, serum amyloid A and serum silicon levels. Ferritin subunit expression in BAL and transbronchial biopsies was analysed by reverse transcription quantitative PCR. Lipid accumulation in BAL macrophages was assessed by Oil Red O staining. RESULTS: Serum iron levels were significantly elevated in patients with silicosis, with a strong positive association with serum ferritin levels. In contrast, markers of systemic inflammation were not increased in silicosis patients. Serum silicon levels were significantly elevated in complicated disease. BAL macrophages from silicosis patients were morphologically consistent with lipid-laden foamy macrophages. Ferritin light chain (FTL) mRNA expression in BAL macrophages was also significantly elevated in simple silicosis patients and correlated with systemic ferritin. CONCLUSION: Our findings suggest that elevated iron levels during the early phases of silicosis increase FTL expression in BAL macrophages, which drives elevated BAL and serum ferritin levels. Excess iron and ferritin were also associated with the emergence of a foamy BAL macrophage phenotype. Ferritin may represent an early disease marker for silicosis, where increased levels are independent of inflammation and may contribute to fibrotic lung remodelling.

Cigarette Smoking Exacerbates Skeletal Muscle Injury without Compromising Its Regenerative Capacity.

Skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease negatively impacts quality of life and survival. Cigarette smoking (CS) is the major risk factor for chronic obstructive pulmonary disease and skeletal muscle dysfunction; however, how CS affects skeletal muscle function remains enigmatic. To examine the impact of CS on skeletal muscle inflammation and regeneration, male BALB/c mice were exposed to CS for 8 weeks before muscle injury was induced by barium chloride injection, and were maintained on the CS protocol for up to 21 days after injury. Barium chloride injection resulted in architectural damage to the tibialis anterior muscle, resulting in a decrease contractile function, which was worsened by CS exposure. CS exposure caused muscle atrophy (reduction in gross weight and myofiber cross-sectional area) and altered fiber type composition (31% reduction of oxidative fibers). Both contractile function and loss in myofiber cross-sectional area by CS exposure gradually recovered over time. Satellite cells are muscle stem cells that confer skeletal muscle the plasticity to adapt to changing demands. CS exposure blunted Pax7+ centralized nuclei within satellite cells and thus prevented the activation of these muscle stem cells. Finally, CS triggered muscle inflammation; in particular, there was an exacerbated recruitment of F4/80+ monocytic cells to the site of injury along with enhanced proinflammatory cytokine expression. In conclusion, CS exposure amplified the local inflammatory response at the site of skeletal muscle injury, and this was associated with impaired satellite cell activation, leading to a worsened muscle injury and contractile function without detectable impacts on the recovery outcomes.

Increased MUC1 plus a larger quantity and complex size for MUC5AC in the peripheral airway lumen of long-term tobacco smokers.

There is little information on mucins versus potential regulatory factors in the peripheral airway lumen of long-term smokers with (LTS+) and without (LTS-) chronic obstructive pulmonary disease (COPD). We explored these matters in bronchoalveolar lavage (BAL) samples from two study materials, both including LTS+ and LTS- with a very similar historic exposure to tobacco smoke, and healthy non-smokers (HNSs; n=4-20/group). Utilizing slot blot and immunodetection of processed (filtered and centrifuged), as well as unprocessed BAL samples from one of the materials, we compared the quantity and fraction of large complexes of mucins. All LTS displayed an enhanced (median) level of MUC5AC compared with HNS. LTS- displayed a higher level of large MUC5AC complexes than HNS while LTS+ displayed a similar trend. In all LTS, total MUC5AC correlated with blood leukocytes, BAL neutrophil elastase and net gelatinase activity. Large mucin complexes accounted for most MUC5B, without clear group differences. In all LTS, total MUC5B correlated with total MUC5AC and local bacteria. In the same groups, large MUC5B complexes correlated with serum cotinine. MUC1 was increased and correlated with BAL leukocytes in all LTS whereas MUC2 was very low and without clear group differences. Thus, the main part of MUC5AC and MUC5B is present as large complexes in the peripheral airway lumen and historic as well as current exposure to tobacco smoke emerge as potential regulatory factors, regardless of COPD per se. Bacteria, leukocytes and proteinases also constitute potential regulatory factors, of interest for future therapeutic strategies.

The role of microglia in the second and third postnatal weeks of life in rat hippocampal development and memory.

Microglia are resident immune cells of the central nervous system (CNS). In adulthood they are involved in surveillance and responses to pathogens and injury and prenatally they play a role in brain development. However, the role of microglia during the early postnatal period and how they impact development long-term remains poorly understood. Here, to investigate the specific role of microglia in postnatal development, we used a Cx3cr1-Dtr transgenic Wistar rat model to acutely ablate microglia from either postnatal day (P) 7 or 14. We specifically assessed how transient microglial ablation affected astrocytes and neurons acutely, during the juvenile period, and in adulthood. Hippocampal microglial numbers remained low at P21 in the P7-ablated animals and complexity remained reduced after P14-ablation. This protracted effect on these key immune cells led to a small but significant increase in CA1 mature neuron numbers and a significant increase in astrocyte density in the subgranular dentate gyrus in adults that had their microglia ablated at P14. However, these histological differences were small, and spatial and recognition memory in novel objection and place recognition tests were not affected. Overall, our data reveal for the first time that the transient depletion of microglia during the neonatal period impacts briefly on the brain but that the long-lasting effects are minimal. Neonatal microglia may be dispensable in the establishment of hippocampal brain function. These data also imply that novel therapeutic anti-inflammatories that cross the blood-brain barrier to inhibit microglia are unlikely to have long-term negative consequences if administered in the neonatal period.

New frontiers in the treatment of comorbid cardiovascular disease in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a disease characterised by persistent airflow limitation that is not fully reversible and is currently the fourth leading cause of death globally. It is now well established that cardiovascular-related comorbidities contribute to morbidity and mortality in COPD, with approximately 50% of deaths in COPD patients attributed to a cardiovascular event (e.g. myocardial infarction). Cardiovascular disease (CVD) and COPD share various risk factors including hypertension, sedentarism, smoking and poor diet but the underlying mechanisms have not been fully established. However, there is emerging and compelling experimental and clinical evidence to show that increased oxidative stress causes pulmonary inflammation and that the spill over of pro-inflammatory mediators from the lungs into the systemic circulation drives a persistent systemic inflammatory response that alters blood vessel structure, through vascular remodelling and arterial stiffness resulting in atherosclerosis. In addition, regulation of endothelial-derived vasoactive substances (e.g. nitric oxide (NO)), which control blood vessel tone are altered by oxidative damage of vascular endothelial cells, thus promoting vascular dysfunction, a key driver of CVD. In this review, the detrimental role of oxidative stress in COPD and comorbid CVD are discussed and we propose that targeting oxidant-dependent mechanisms represents a novel strategy in the treatment of COPD-associated CVD.

Matrine reduces cigarette smoke-induced airway neutrophilic inflammation by enhancing neutrophil apoptosis.

Chronic Obstructive Pulmonary Disease (COPD) is a major incurable global health burden and will become the third largest cause of death in the world by 2030. It is well established that an exaggerated inflammatory and oxidative stress response to cigarette smoke (CS) leads to, emphysema, small airway fibrosis, mucus hypersecretion, and progressive airflow limitation. Current treatments have limited efficacy in inhibiting chronic inflammation and consequently do not reverse the pathology that initiates and drives the long-term progression of disease. In particular, there are no effective therapeutics that target neutrophilic inflammation in COPD, which is known to cause tissue damage by degranulation of a suite of proteolytic enzymes including neutrophil elastase (NE). Matrine, an alkaloid compound extracted from Sophora flavescens Ait, has well known anti-inflammatory activity. Therefore, the aim of the present study was to investigate whether matrine could inhibit CS-induced lung inflammation in mice. Matrine significantly reduced CS-induced bronchoalveolar lavage fluid (BALF) neutrophilia and NE activity in mice. The reduction in BALF neutrophils in CS-exposed mice by matrine was not due to reductions in pro-neutrophil cytokines/chemokines, but rather matrine's ability to cause apoptosis of neutrophils, which we demonstrated ex vivo Thus, our data suggest that matrine has anti-inflammatory actions that could be of therapeutic potential in treating CS-induced lung inflammation observed in COPD.

Increased hypothalamic microglial activation after viral-induced pneumococcal lung infection is associated with excess serum amyloid A production.

BACKGROUND: It is well established that lung pathology and inflammation are more severe during respiratory infections complicated by the presence of both bacteria and viruses. Whilst co-infection can result in invasive pneumococcal disease and systemic inflammation, the neuroinflammatory consequences of co-infection are poorly characterised. METHODS: In this study, we utilised a mouse co-infection model involving Streptococcus pneumoniae (S. pneumoniae) and influenza A virus (IAV) lung infection, and we also isolated microglia for ex vivo stimulation with pneumococcus or serum amyloid A (SAA). RESULTS: Co-infection but not S. pneumoniae or IAV alone significantly increased the number of amoeboid-shaped microglia and expression of pro-inflammatory cytokines including tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and C-C motif chemokine ligand-2 (CCL-2) in the hypothalamus. Pneumococcus was only detected in the hypothalamus of co-infected mice. In addition, the systemic inflammatory cytokines TNFα, IL-1ß and IL-6 were not elevated in co-infected mice relative to IAV-infected mice, whereas SAA levels were markedly increased in co-infected mice (p < 0.05). SAA and its functional receptor termed formyl peptide receptor 2 (Fpr2) transcript expression were also increased in the hypothalamus. In mouse primary microglia, recombinant SAA but not S. pneumoniae stimulated TNFα, IL-1ß, IL-6 and CCL-2 expression, and this response was completely blocked by the pro-resolving Fpr2 agonist aspirin-triggered resolvin D1 (AT-RvD1). CONCLUSIONS: In summary, lung co-infection increased the number of 'activated' amoeboid-shaped microglia and inflammatory cytokine expression in the hypothalamus. Whilst persistent pneumococcal brain infection was observed, SAA proved to be a much more potent stimulus of microglia than pneumococci, and this response was potently suppressed by the anti-inflammatory AT-RvD1. Targeting Fpr2 with pro-resolving eicosanoids such as AT-RvD1 may restore microglial homeostasis during severe respiratory infections.

Tumour-associated neutrophils and loss of epithelial PTEN can promote corticosteroid-insensitive MMP-9 expression in the chronically inflamed lung microenvironment.

Matrix metalloproteinase-9 (MMP-9) is increased in a number of pathological lung conditions, where the proteinase contributes to deleterious remodelling of the airways. While both lung cancer and COPD are associated with increased MMP-9 expression, the cellular and molecular drivers of MMP-9 remain unresolved. In this study, MMP-9 transcript measured within the tumour region from patients with non-small-cell lung cancer (NSCLC) and coexisting COPD was found to be uniformly increased relative to adjacent tumour-free tissue. MMP-9 gene expression and immunohistochemistry identified tumour-associated neutrophils, but not macrophages, as a predominant source of this proteinase. In addition, PTEN gene expression was significantly reduced in tumour and there was evidence of epithelial MMP-9 expression. To explore whether PTEN can regulate epithelial MMP-9 expression, a small interfering (si)RNA knockdown strategy was used in Beas-2B bronchial epithelial cells. PTEN knockdown by siRNA selectively increased MMP-9 expression in response to lipopolysaccharide in a corticosteroid-insensitive manner. In summary, tumour-associated neutrophils represent an important source of MMP-9 in NSCLC, and loss of epithelial PTEN may further augment steroid-insensitive expression.

Aspirin-triggered resolvin D1 reduces pneumococcal lung infection and inflammation in a viral and bacterial coinfection pneumonia model.

Formyl peptide receptor 2/lipoxin A4 (LXA4) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs.