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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.

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease. Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.

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+D(b)NP(366)+ and D(b)PA(224)+ 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.

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology. The common cytokine binding site of the human common beta (β c ) receptor signals for three inflammatory cytokines: GM-CSF, IL-5 and IL-3. In this study, β c was targeted with the monoclonal antibody (mAb) CSL311 in engineered mice devoid of mouse β c and β IL-3 and expressing human β c (hβ c Tg mice). Direct pulmonary administration of lipopolysaccharide (LPS) caused ARDS-like lung injury, and CSL311 markedly reduced lung inflammation and oedema, resulting in improved oxygen saturation levels in hβ c Tg mice. In a separate model, influenza (HKx31) lung infection caused viral pneumonia associated with a large influx of myeloid cells into the lungs of hβ c Tg mice. The therapeutic application of CSL311 potently decreased accumulation of monocytes/macrophages, neutrophils, and eosinophils without altering lung viral loads. Furthermore, CSL311 treatment did not limit the viral-induced expansion of NK and NKT cells, or the tissue expression of type I/II/III interferons needed for efficient viral clearance. Simultaneously blocking GM-CSF, IL-5 and IL-3 signalling with CSL311 may represent an improved and clinically applicable strategy to reducing hyperinflammation in the ARDS setting.

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.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient‐derived) Kras G12D ‐driven LAC models, the specific blockade of ADAM17, including with a non‐toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro‐tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL‐6R, to release soluble IL‐6R that drives IL‐6 trans‐signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in Kras G12D ‐driven LAC was independent of bone marrow‐derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho‐ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS ‐driven LAC and provide the rationale to employ ADAM17‐based therapeutic strategies for targeting KRAS mutant cancers. Synopsis Oncogenic KRAS mutations are associated with one‐third of lung adenocarcinoma (LAC) cases, yet the downstream molecular events that facilitate KRAS‐mediated tumorigenesis in the lung remain unresolved. This study reveals an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. The specific genetic and therapeutic targeting of ADAM17, the latter with a non‐toxic prodomain inhibitor, suppressed tumour burden by reducing cellular proliferation in genetically‐engineered and xenograft mutant KRAS‐driven LAC models. The pro‐tumorigenic activity of ADAM17 in LAC was dependent upon its threonine phosphorylation by p38 MAPK. ADAM17 preferentially shed the substrate, IL‐6R, during LAC to release soluble IL‐6R that drives IL‐6 trans‐signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in mutant KRAS‐driven LAC was independent of bone marrow‐derived hematopoietic immune cells. In KRAS mutant human LAC, augmented phospho‐ADAM17 levels were observed primarily in epithelial rather than immune cells, and significantly and positively correlated with activated p38 and ERK1/2 MAPK pathways. Graphical Abstract Oncogenic KRAS mutations are associated with one‐third of lung adenocarcinoma (LAC) cases, yet the downstream molecular events that facilitate KRAS‐mediated tumorigenesis in the lung remain unresolved. This study reveals an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC.

Chronic obstructive pulmonary disease (COPD) will soon be the third most common cause of death globally. Despite smoking cessation, neutrophilic mucosal inflammation persistently damages the airways and fails to protect from recurrent infections. This maladaptive and excess inflammation is also refractory to glucocorticosteroids (GC). Here, we identify serum amyloid A (SAA) as a candidate mediator of GC refractory inflammation in COPD. Extrahepatic SAA was detected locally in COPD bronchoalveolar lavage fluid, which correlated with IL-8 and neutrophil elastase, consistent with neutrophil recruitment and activation. Immunohistochemistry detected SAA was in close proximity to airway epithelium, and in vitro SAA triggered release of IL-8 and other proinflammatory mediators by airway epithelial cells in an ALX/FPR2 (formyl peptide receptor 2) receptor-dependent manner. Lipoxin A 4 (LXA 4 ) can also interact with ALX/FPR2 receptors and lead to allosteric inhibition of SAA-initiated epithelial responses (pA 2 13 nM). During acute exacerbation, peripheral blood SAA levels increased dramatically and were disproportionately increased relative to LXA 4 . Human lung macrophages (CD68 + ) colocalized with SAA and GCs markedly increased SAA in vitro (THP-1, pEC 50 43 nM). To determine its direct actions, SAA was administered into murine lung, leading to induction of CXC chemokine ligand 1/2 and a neutrophilic response that was inhibited by 15-epi-LXA 4 but not dexamethasone. Taken together, these findings identify SAA as a therapeutic target for inhibition and implicate SAA as a mediator of GC-resistant lung inflammation that can overwhelm organ protective signaling by lipoxins at ALX/FPR2 receptors.

Much of the total disease burden and cost of chronic obstructive pulmonary disease (COPD) is associated with acute exacerbations of COPD (AECOPD). Serum amyloid A (SAA) is a novel candidate exacerbation biomarker identified by proteomic screening. To assess SAA as a biomarker of AECOPD. Biomarkers were assessed (1) cross-sectionally (stable vs. AECOPD; 62 individuals) and (2) longitudinally with repeated measures (baseline vs. AECOPD vs. convalescence; 78 episodes in 37 individuals). Event severity was graded (I, ambulatory; II, hospitalized; III, respiratory failure) based on consensus guidelines. Presumptively newly acquired pathogens were associated with onset of symptomatic AECOPD. In the cross-sectional study, both SAA and C-reactive protein (CRP) were elevated at AECOPD onset compared with stable disease (SAA median, 7.7 vs. 57.6 mg/L; P < 0.01; CRP median, 4.6 vs. 12.5 mg/L; P < 0.01). Receiver operator characteristics analysis was used to generate area-under-curve values for event severity. SAA discriminated level II/III events (SAA, 0.88; 95% confidence interval, 0.80-0.94 vs. CRP, 0.80; 95% confidence interval, 0.70-0.87; P = 0.05). Combining SAA or CRP with major symptoms (Anthonisen criteria, dyspnea) did not further improve the prediction model for severe episodes. IL-6 and procalcitonin were not informative. SAA is a novel blood biomarker of AECOPD that is more sensitive than CRP alone or in combination with dyspnea. SAA may offer new insights into the pathogenesis of AECOPD.

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 Ly6C low patrolling monocytes and Ly6C high 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.

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-1β (IL-1β), 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-1β 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-1β, 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.