The innate immune inflammatory response to lipopolysaccharide (LPS, an endotoxin) is essential for lung host defense against infection by gram-negative bacteria but is also implicated in the pathogenesis of some lung diseases. Studies on genetically altered mice implicate granulocyte-macrophage colony-stimulating factor (GM-CSF) in lung responses to LPS; however, the physiological effects of GM-CSF neutralization are poorly characterized. We performed detailed kinetic and dose-response analyses of the lung inflammation response to LPS in the presence of the specific GM-CSF-neutralizing antibody 22E9. LPS instilled into the lungs of BALB/c mice induced a dose-dependent inflammation comprised of intense neutrophilia, macrophage infiltration and proliferation, TNF-alpha and matrix metalloproteinase release, and macrophage inflammatory protein-2 induction. The neutralization of anti-GM-CSF in a dose-dependent fashion suppressed these inflammatory indexes by 85% when given before or after LPS or after repeat LPS challenges. Here we report for the first time that the physiological expression of Toll-like receptor-4 in lung is reduced by anti-GM-CSF. We observed that lower Toll-like receptor-4 expression correlated with a similar decline in peak TNF- levels in response to endotoxin. Consequently, sustained expression of key inflammatory mediators over 24 h was reduced. These data expand the understanding of the contribution of GM-CSF to innate immune responses in lung and suggest that blocking GM-CSF might benefit some lung diseases where LPS has been implicated in etiology.

Background and ObjectiveNeurocognitive dysfunction is present in up to ∼61% of people with chronic obstructive pulmonary disease (COPD), with symptoms including learning and memory deficiencies, negatively impacting the quality of life of these individuals. As the mechanisms responsible for neurocognitive deficits in COPD remain unknown, we explored whether chronic cigarette smoke (CS) exposure causes neurocognitive dysfunction in mice and whether this is associated with neuroinflammation and an altered neuropathology.MethodsMale BALB/c mice were exposed to room air (sham) or CS (9 cigarettes/day, 5 days/week) for 24 weeks. After 23 weeks, mice underwent neurocognitive tests to assess working and spatial memory retention. At 24 weeks, mice were culled and lungs were collected and assessed for hallmark features of COPD. Serum was assessed for systemic inflammation and the hippocampus was collected for neuroinflammatory and structural analysis.ResultsChronic CS exposure impaired lung function as well as driving pulmonary inflammation, emphysema, and systemic inflammation. CS exposure impaired working memory retention, which was associated with a suppression in hippocampal microglial number, however, these microglia displayed a more activated morphology. CS-exposed mice showed changes in astrocyte density as well as a reduction in synaptophysin and dendritic spines in the hippocampus.ConclusionWe have developed an experimental model of COPD in mice that recapitulates the hallmark features of the human disease. The altered microglial/astrocytic profiles and alterations in the neuropathology within the hippocampus may explain the neurocognitive dysfunction observed during COPD.

Background and Purpose: It is well established that both smokers and patients with COPD are at a significantly heightened risk of cardiovascular disease (CVD), although the mechanisms underpinning the onset and progression of comorbid CVD are largely unknown. Here, we explored whether cigarette smoke (CS) exposure impairs vascular function in mice and given the well-known pathological role for oxidative stress in COPD, whether the antioxidant compound ebselen prevents CS-induced vascular dysfunction in mice. Experimental Approach: Male BALB/c mice were exposed to either room air (sham) or CS generated from 9 cigarettes per day, 5 days a week for 8 weeks. Mice were treated with ebselen (10mg/kg, oral gavage once daily) or vehicle (5% w/v CM cellulose in water) 1 h prior to the first CS exposure of the day. Upon sacrifice, bronchoalveolar lavage fluid (BALF) was collected to assess pulmonary inflammation and the thoracic aorta was excised to investigate vascular endothelial and smooth muscle dilator responses ex-vivo. Key Results: CS exposure caused a significant increase in lung inflammation which was reduced by ebselen. CS also caused significant endothelial dysfunction in the thoracic aorta which was attributed to a downregulation of eNOS expression and increased vascular oxidative stress. Ebselen abolished the aortic endothelial dysfunction seen in CS-exposed mice by reducing the oxidative burden and preserving eNOS expression. Conclusion and Implications: Targeting CS-induced oxidative stress with ebselen may provide a novel means for treating the life-threatening pulmonary and cardiovascular manifestations associated with cigarette smoking and COPD.

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Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr4−/− mice by 6 months of age, the lungs of Tlr2−/− mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr4−/− mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal−/− mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal−/− mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr4−/− mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema.

Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-β expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-β response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-β increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.

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.

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.