Apocynin and ebselen reduce influenza A virus-induced lung inflammation in cigarette smoke-exposed mice

Influenza A virus (IAV) infections are a common cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Oxidative stress is increased in COPD, IAV-induced lung inflammation and AECOPD. Therefore, we investigated whether targeting oxidative stress with the Nox2 oxidase inhibitors and ROS scavengers, apocynin and ebselen could ameliorate lung inflammation in a mouse model of AECOPD. Male BALB/c mice were exposed to cigarette smoke (CS) generated from 9 cigarettes per day for 4 days. On day 5, mice were infected with 1 × 104.5 PFUs of the IAV Mem71 (H3N1). BALF inflammation, viral titers, superoxide production and whole lung cytokine, chemokine and protease mRNA expression were assessed 3 and 7 days post infection. IAV infection resulted in a greater increase in BALF inflammation in mice that had been exposed to CS compared to non-smoking mice. This increase in BALF inflammation in CS-exposed mice caused by IAV infection was associated with elevated gene expression of pro-inflammatory cytokines, chemokines and proteases, compared to CS alone mice. Apocynin and ebselen significantly reduced the exacerbated BALF inflammation and pro-inflammatory cytokine, chemokine and protease expression caused by IAV infection in CS mice. Targeting oxidative stress using apocynin and ebselen reduces IAV-induced lung inflammation in CS-exposed mice and may be therapeutically exploited to alleviate AECOPD

Nox1 oxidase suppresses influenza a virus-induced lung inflammation and oxidative stress

Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1−/y) mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×104 PFU) influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1−/y mice resulted in significantly greater: loss of bodyweight (Day 3); BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1−/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1−/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8+ and CD4+ T lymphocytes, and of Tregs were similar between WT and Nox1−/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear to have opposing roles in the regulation of inflammation caused by influenza A viruses

Rhenium(I) conjugates as tools for tracking cholesterol in cells

Cholesterol is vital to control membrane integrity and fluidity, but is also a precursor to produce steroid hormones, bile acids, and vitamin D. Consequently, altered cholesterol biology has been linked to many diseases, including metabolic syndromes and cancer. Defining the intracellular pools of cholesterol and its trafficking within cells is essential to understand both normal cell physiology and mechanisms of pathogenesis. We have synthesized a new cholesterol mimic (ReTEGCholestanol), comprising a luminescent rhenium metal complex and a cholestanol targeting unit, linked using a tetraethylene glycol (TEG) spacer. ReTEGCholestanol demonstrated favourable imaging properties and improved water solubility when compared to a cholesterol derivative, and structurally related probes lacking the TEG linker. A non-malignant and three malignant prostate cell lines were used to characterize the uptake and intracellular distribution of ReTEGCholestanol. The ReTEGCholestanol complex was effectively internalized and mainly localized to late endosomes/lysosomes in non-malignant PNT1a cells, while in prostate cancer cells it also accumulated in early endosomes and multivesicular bodies, suggesting disturbed cholesterol biology in the malignant cells. The ReTEGCholestanol is a novel imaging agent for visualizing endosomal uptake and trafficking, which may be used to define cholesterol related biology including membrane integration and altered lipid trafficking/processing

Gestational influenza A virus infection elicits nonresolving vascular dysfunction and T-cell accumulation in the aorta of mice.

T-cell accumulation within the aorta promotes endothelial dysfunction and the genesis of cardiovascular disease, including hypertension and atherosclerosis. Viral infection during pregnancy is also known to mediate marked acute endothelial dysfunction, but it is not clear whether T cells are recruited to the aorta and whether the dysfunction persists postpartum. Here, we demonstrate that influenza A virus (IAV) infection during pregnancy in a murine model resulted in endothelial dysfunction of the aorta, which persisted for up to 60 days postinfection and was associated with higher levels of IFN-γ mRNA expression within the tissue. In the absence of infection, low numbers of naïve CD4+ and CD8+ T cells, central memory T cells, and effector memory T cells were observed in the aorta. However, with IAV infection, these T-cell subsets were significantly increased with a notable accumulation of IAV-specific CD8+ effector memory T cells. Critically, this increase was maintained out to at least 60 days. In contrast, IAV infection in nonpregnant female mice resulted in modest endothelial dysfunction with no accumulation of T cells within the aorta. These data, therefore, demonstrate that the aorta is a site of T-cell recruitment and retention after IAV infection during pregnancy. Although IAV-specific memory T cells could theoretically confer protection against future influenza infection, nonspecific memory T-cell activation and IFN-γ production in the aorta could also contribute to future endothelial dysfunction and cardiovascular disease.NEW & NOTEWORTHY Pregnancy is a risk factor for cardiovascular complications to influenza A virus (IAV) infection. We demonstrate that gestational IAV infection caused endothelial dysfunction of the maternal aorta, which persisted for 60 days postinfection in mice. Various T cells accumulated within the aorta at 60 days because of the infection, and this was associated with elevated levels of the proinflammatory cytokine, IFN-γ. Our study demonstrates a novel "long influenza" cardiovascular phenotype in female mice

Low dose aspirin prevents endothelial dysfunction in the aorta and foetal loss in pregnant mice infected with influenza A virus.

Influenza A virus (IAV) infection in pregnancy resembles a preeclamptic phenotype characterised by vascular dysfunction and foetal growth retardation. Given that low dose aspirin (ASA) is safe in pregnancy and is used to prevent preeclampsia, we investigated whether ASA or NO-conjugated aspirin, NCX4016, resolve vascular inflammation and function to improve offspring outcomes following IAV infection in pregnant mice. Pregnant mice were intranasally infected with a mouse adapted IAV strain (Hkx31; 104 plaque forming units) and received daily treatments with either 200µg/kg ASA or NCX4016 via oral gavage. Mice were then culled and the maternal lungs and aortas collected for qPCR analysis, and wire myography was performed on aortic rings to assess endothelial and vascular smooth muscle functionality. Pup and placentas were weighed and pup growth rates and survival assessed. IAV infected mice had an impaired endothelial dependent relaxation response to ACh in the aorta, which was prevented by ASA and NCX4016 treatment. ASA and NCX4016 treatment prevented IAV dissemination and inflammation of the aorta as well as improving the pup placental ratios in utero, survival and growth rates at post-natal day 5. Low dose ASA is safe to use during pregnancy for preeclampsia and this study demonstrates that ASA may prove a promising treatment for averting the significant vascular complications associated with influenza infection during pregnancy

Brain region-specific alterations in gene expression trajectories in the offspring born from influenza A virus infected mice.

Influenza A virus (IAV) infection during pregnancy can increase the risk for neurodevelopmental disorders in the offspring, however, the underlying neurobiological mechanisms are largely unknown. To recapitulate viral infection, preclinical studies have traditionally focused on using synthetic viral mimetics, rather than live IAV, to examine consequences of maternal immune activation (MIA)-dependent processes on offspring. In contrast, few studies have used live IAV to assess effects on global gene expression, and none to date have addressed whether moderate IAV, mimicking seasonal influenza disease, alters normal gene expression trajectories in different brain regions across different stages of development. Herein, we show that moderate IAV infection during pregnancy, which causes mild maternal disease and no overt foetal complications in utero, induces lasting effects on the offspring into adulthood. We observed behavioural changes in adult offspring, including disrupted prepulse inhibition, dopaminergic hyper-responsiveness, and spatial recognition memory deficits. Gene profiling in the offspring brain from neonate to adolescence revealed persistent alterations to normal gene expression trajectories in the prefronal cortex, hippocampus, hypothalamus and cerebellum. Alterations were found in genes involved in inflammation and neurogenesis, which were predominately dysregulated in neonatal and early adolescent offspring. Notably, late adolescent offspring born from IAV infected mice displayed altered microglial morphology in the hippocampus. In conclusion, we show that moderate IAV during pregnancy perturbs neurodevelopmental trajectories in the offspring, including alterations in the neuroinflammatory gene expression profile and microglial number and morphology in the hippocampus, resulting in behavioural changes in adult offspring. Such early perturbations may underlie the vulnerability in human offspring for the later development of neurodevelopmental disorders, including schizophrenia. Our work highlights the importance of using live IAV in developing novel preclinical models that better recapitulate the real-world impact of inflammatory insults during pregnancy on offspring neurodevelopmental trajectories and disease susceptibility later in life

CDKI-73 is a novel pharmacological inhibitor of Rab11 cargo delivery and innate immune secretion

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.Alexandra Sorvina, Tetyana Shandala, Shudong Wang, David J. Sharkey, Emma Parkinson-Lawrence, Stavros Selemidis and Douglas A. Brook

Inhibition of Nox2 Oxidase Activity Ameliorates Influenza A Virus-Induced Lung Inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8+ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2−/y mice. The numbers of influenza-specific CD8+DbNP366+ and DbPA224+ T cells in the BALF and spleen were comparable in WT and Nox2−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner

Nontypeable haemophilus influenzae induces sustained lung oxidative stress and protease expression

© 2015 King et al. Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps

A paradigm in immunochemistry, revealed by monoclonal antibodies to spatially distinct epitopes on Syntenin-1

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells.Ian R. D. Johnson, Alexandra Sorvina, Jessica M. Logan, Courtney R. Moore, Jessica K. Heatlie, Emma J. Parkinson-Lawrence, Stavros Selemidis, John J. O’Leary, Lisa M. Butler and Douglas A. Brook