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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.

Respiratory syncytial virus (RSV) commonly infects the upper respiratory tract (URT) of humans, manifesting with mild cold or flu-like symptoms. However, in infants and the elderly, severe disease of the lower respiratory tract (LRT) often occurs and can develop into chronic airway disease. A better understanding of how an acute RSV infection transitions to a LRT chronic inflammatory disease is critically important to improve patient care and long-term health outcomes. To model acute and chronic phases of the disease, we infected wild-type C57BL/6 and toll-like receptor 7 knockout (TLR7 KO) mice with RSV and temporally assessed nasal, airway and lung inflammation for up to 42 days post-infection. We show that TLR7 reduced viral titers in the URT during acute infection but promoted pronounced pathogenic and chronic airway inflammation and hyperreactivity in the LRT. This study defines a hitherto unappreciated molecular mechanism of lower respiratory pathogenesis to RSV, highlighting the potential of TLR7 modulation to constrain RSV pathology to the URT.

Endosomal NOX2 oxidase-dependent ROS production promotes influenza pathogenicity, but the role of NOX4 oxidase, which is highly expressed in the lung endothelium, is largely unknown. The aim of this study was to determine if endothelial NOX4 expression can influence viral pathology in vivo, using a mouse model of influenza infection. WT and transgenic endothelial NOX4 overexpressing mice (NOX4 TG) were infected intranasally with the Hong Kong H3N2 X-31 influenza A virus (104 PFU; HK x-31) or PBS control. Mice were culled at either 3 or 7 days post-infection to analyse: airway inflammation by bronchoalveolar lavage fluid (BALF) cell counts; NOX4, as well as inflammatory cytokine and chemokine gene expression by QPCR; and ROS production by an L-012-enhanced chemiluminescence assay. Influenza A virus infection of WT mice resulted in a significant reduction in lung NOX4 mRNA at day 3, which persisted until day 7, when compared to uninfected mice. Influenza A virus infection of NOX4 TG mice resulted in significantly less weight loss than that of WT mice at 3-days post infection. Viral titres were decreased in infected NOX4 TG mice compared to the infected WT mice, at both 3- and 7-days post infection and there was significantly less lung alveolitis, peri-bronchial inflammation and neutrophil infiltration. The oxidative burst from BALF inflammatory cells extracted from infected NOX4 TG mice was significantly less than that in the WT mice. Expression of macrophage and neutrophil chemoattractants CXCL10, CCL3, CXCL1 and CXCL2 in the lung tissue were significantly lower in NOX4 TG mice compared to the WT mice at 3-days post infection. We conclude that endothelial NOX4 oxidase is protective against influenza morbidity and is a potential target for limiting influenza A virus-induced lung inflammation.

There is an urgent need to develop effective therapeutic strategies including immunomodulators to combat influenza A virus (IAV) infection. Influenza A viruses increase ROS production, which suppress anti-viral responses and contribute to pathological inflammation and morbidity. Two major cellular sites of ROS production are endosomes via the NOX2-oxidase enzyme and the electron transport chain in mitochondria. Here we examined the effect of administration of Cgp91ds-TAT, an endosome-targeted NOX2 oxidase inhibitor, in combination with mitoTEMPO, a mitochondrial ROS scavenger and compared it to monotherapy treatment during an established IAV infection. Mice were infected with IAV (Hkx31 strain; 104PFU/mouse) and 24 h post infection were treated with Cgp91ds-TAT (0.2 mg/kg), mitoTEMPO (100 μg) or with a combination of these inhibitors [Cgp91ds-TAT (0.2 mg/kg)/mitoTEMPO (100 μg)] intranasally every day for up to 2 days post infection (pi). Mice were euthanized on Days 3 or 6 post infection for analyses of disease severity. A combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than the ROS inhibitors alone at reducing airway and neutrophilic inflammation, bodyweight loss, lung oedema and improved the lung pathology with a reduction in alveolitis following IAV infection. Dual ROS inhibition also caused a significant elevation in Type I IFN expression at the early phase of infection (day 3 pi), however, this response was suppressed at the later phase of infection (day 6 pi). Furthermore, combined treatment with Cgp91ds-TAT and mitoTEMPO resulted in an increase in IAV-specific CD8+ T cells in the lungs. In conclusion, this study demonstrates that the reduction of ROS production in two major subcellular sites, i.e. endosomes and mitochondria, by intranasal delivery of a combination of Cgp91ds-TAT and mitoTEMPO, suppresses the severity of influenza infection and highlights a novel immunomodulatory approach for IAV disease management.

AimsAnimal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart.MethodsWe used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at 105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI.Results73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation.ConclusionThere were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart.

Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage.Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction.Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep.Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction.