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+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.     

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.     

A Co-Culture System with an Organotypic Lung Slice and an Immortal Alveolar Macrophage Cell Line to Quantify Silica-Induced Inflammation

There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages) of wild type and P2rx7^−/− mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7^−/− mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7^−/− mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7^−/− mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

8-Oxoguanine-DNA glycosylase 1 deficiency modifies allergic airway inflammation by regulating STAT6 and IL-4 in cells and in mice

8-Oxoguanine-DNA glycosylase (OGG-1) is a base excision DNA repair enzyme; however, its function in modulating allergic diseases remains undefined. Using OGG-1 knockout (KO) mice, we show that this protein affects allergic airway inflammation after sensitization and challenge by ovalbumin(OVA). OGG-1 KO mice exhibited less inflammatory cell infiltration and reduced oxidative stress in the lungs after OVA challenge compared to WT mice. The KO phenotype included decreased IL-4, IL-6, IL-10, and IL-17 in lung tissues. In addition, OGG-1 KO mice showed decreased expression and phosphorylation of STAT6 as well as NF-κB. Down-regulation of OGG-1 by siRNA lowered ROS and IL-4 levels but increased IFN-γ production in cultured epithelial cells after exposure to house dust mite extracts. OGG-1 may affect the levels of oxidative stress and proinflammatory cytokines during asthmatic conditions. OGG-1 deficiency negatively regulates allergen-induced airway inflammatory response.     

Epithelial Cell Mitochondrial Dysfunction and PINK1 Are Induced by Transforming Growth Factor- Beta1 in Pulmonary Fibrosis

Background

Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy) in alveolar epithelial cell death and fibrosis.

Methods

We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1), in IPF lung tissues by Western blotting, transmission electron microscopy (TEM), and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1). Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis.

Results

Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1 ^-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis.

Conclusion

TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.     

Loss of Gadd45a does not modify the pulmonary response to oxidative stress

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.     

Estrogen receptor-alpha regulates pulmonary alveolar loss and regeneration in female mice: morphometric and gene expression studies

Pulmonary alveoli, especially in females, are estrogen-responsive structures: ovariectomy in wild-type (WT) adult mice results in alveolar loss, and estradiol replacement induces alveolar regeneration. Furthermore, estrogen receptor (ER)-alpha and ER-beta are required for the developmental formation of a full complement of alveoli in female mice. We now show ovariectomy resulted in alveolar loss in adult ER-beta(-/-) mice but not in adult ER-alpha(-/-) mice. Estradiol treatment of ovariectomized ER-beta(-/-) mice induced alveolar regeneration. In ovariectomized WT mice, estradiol treatment resulted, within 1 h, in RNA-level gene expression supportive of processes needed to form an alveolar septum, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Among these processes, protein expression supporting angiogenesis and cell replication was elevated 1 and 3 h, respectively, after estradiol treatment; similar findings were not present in either mutant. We conclude: 1) loss of signaling via ER-beta is not required for postovariectomy-induced alveolar loss or estradiol-induced regeneration; this indicates ER-alpha is key for estrogen-related alveolar loss and regeneration in adult female mice; 2) taken together with prior work showing that developmental formation of a full complement of alveoli requires ER-alpha and ER-beta, the present findings indicate the developmental and regenerative formation of alveoli are regulated differently, i.e., signaling for alveolar regeneration is not merely a recapitulation of signaling for developmental alveologenesis; and 3) the timing of estradiol-induced gene expression in lung supportive of processes required to form a septum differs between ovariectomized WT and ER-beta(-/-) mice.     

TLR2-mediated production of IL-27 and chemokines by respiratory epithelial cells promotes bleomycin-induced pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis is a fatal disease characterized by progressive destruction of the lung. Although TLR2 bridges innate and adaptive immunity by sensing tissue damage, its role in pulmonary fibrosis remains unclear. To address this issue, TLR2(-/-) and WT mice were examined for bleomycin-induced pulmonary fibrosis (BIPF). Flow cytometric and immunohistochemical analysis revealed that TLR2 expression in bronchial epithelial and immune cells of the lungs was upregulated in WT mice during BIPF. Levels of IL-27, TGF-β, chemokines, and hydroxyproline were lower in lungs of TLR2(-/-) mice than in those of WT mice, but IL-17 levels were higher in TLR2(-/-) mice. In in vivo experiments using bone marrow-chimeric mice, TLR2 expression on respiratory epithelial cells, rather than immune cells, induced IL-27 and chemokine production in the lungs, further stimulating BIPF. This effect of TLR2 depended on IRF complexes and MyD88. BIPF was more severe in IL-17A(-/-) mice and in TLR2(-/-) mice treated with anti-IL-17 mAb than in TLR2(-/-) and WT mice. Furthermore, IL-27 blockade in WT mice reduced hydroxyproline levels by enhancing IL-17 production, whereas the treatment of TLR2(-/-) mice with a chemokine mixture increased hydroxyproline levels by recruiting inflammatory cells into the lungs. TLR2 signaling promotes BIPF by inducing IL-27 and chemokine production by respiratory epithelial cells, thereby inhibiting IL-17 production and recruiting inflammatory cells into the lungs.     

MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0272-9) contains supplementary material, which is available to authorized users.     

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.     

Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis

CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung.     

IL10 deficiency promotes alveolar enlargement and lymphoid dysmorphogenesis in the aged murine lung

The connection between aging‐related immune dysfunction and the lung manifestations of aging is poorly understood. A detailed characterization of the aging IL10‐deficient murine lung, a model of accelerated aging and frailty, reconciles features of both immunosenescence and lung aging in a coherent model. Airspace enlargement developed in the middle‐aged (12 months old) and aged (20–22 months old) IL10‐deficient lung punctuated by an expansion of macrophages and alveolar cell apoptosis. Compared to wild‐type (WT) controls, the IL10‐deficient lungs from young (4‐month‐old) mice showed increased oxidative stress which was enhanced in both genotypes by aging. Active caspase 3 staining was increased in the alveolar epithelial cells of aged WT and mutant lungs but was greater in the IL10‐deficient milieu. Lung macrophages were increased in the aged IL10‐deficient lungs with exuberant expression of MMP12. IL10 treatment of naïve and M2‐polarized bone marrow‐derived WT macrophages reduced MMP12 expression. Conditioned media studies demonstrated the secretome of aged mutant macrophages harbors reduced AECII prosurvival factors, specifically keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), promotes cell death, and reduces survival of primary alveolar epithelial cells. Compared to WT controls, aged IL10‐deficient mice have increased parenchymal lymphoid collections comprised of a reduced number of apoptotic cells and B cells. We establish that IL10 is a key modulator of airspace homeostasis and lymphoid morphogenesis in the aging lung enabling macrophage‐mediated alveolar epithelial cell survival and B‐cell survival within tertiary lymphoid structures.     

Role of IL-6 in systemic angiogenesis of the lung.

The multifunctional cytokine interleukin (IL)-6 has been shown to modulate inflammation and angiogenesis. In a mouse model of lung angiogenesis induced by chronic left pulmonary artery ligation (LPAL), we previously showed increased expression of IL-6 mRNA in lung homogenates 4 h after the onset of pulmonary ischemia. To determine whether IL-6 influences both new vessel growth and inflammatory cell influx, we studied wild-type (WT) and IL-6-deficient C57Bl/6J (KO) mice after LPAL (4 h and 1, 7, 14 days). We measured IL-6 protein of the lung by ELISA, the lavage cell profile of the left lung, and new systemic vessel growth with radiolabeled microspheres (14 days after LPAL) in WT and KO mice. We confirmed a 2.4-fold increase in IL-6 protein in the left lung of WT mice compared with right lung 4 h after LPAL. A significant increase in lavaged neutrophils (7.5% of total cells) was observed only in WT mice 4 h after LPAL. New vessel growth was significantly attenuated in KO relative to WT (0.7 vs. 1.9% cardiac output). In an additional series, treatment of WT mice with anti-neutrophil antibody demonstrated a reduction in lavaged neutrophils 4 h after LPAL; however, IL-6 protein remained elevated and neovascularization to the left lung (2.3% cardiac output) was not altered. These results demonstrate that IL-6 plays an important modulatory role in lung angiogenesis, but the changes are not dependent on trapped neutrophils.     

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48 h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24 h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.     

Role of Mincle in Alveolar Macrophage-Dependent Innate Immunity against Mycobacterial Infections in Mice

The role of macrophage-inducible C-type lectin Mincle in lung innate immunity against mycobacterial infection is incompletely defined. In this study, we show that wild-type (WT) mice responded with a delayed Mincle induction on resident alveolar macrophages and newly immigrating exudate macrophages to infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), peaking by days 14-21 posttreatment. As compared with WT mice, Mincle knockout (KO) mice exhibited decreased proinflammatory mediator responses and leukocyte recruitment upon M. bovis BCG challenge, and they demonstrated increased mycobacterial loads in pulmonary and extrapulmonary organ systems. Secondary mycobacterial infection on day 14 after primary BCG challenge led to increased cytokine gene expression in sorted alveolar macrophages of WT mice, but not Mincle KO mice, resulting in substantially reduced alveolar neutrophil recruitment and increased mycobacterial loads in the lungs of Mincle KO mice. Collectively, these data show that WT mice respond with a relatively late Mincle expression on lung sentinel cells to M. bovis BCG infection. Moreover, M. bovis BCG-induced upregulation of C-type lectin Mincle on professional phagocytes critically shapes antimycobacterial responses in both pulmonary and extrapulmonary organ systems of mice, which may be important for elucidating the role of Mincle in the control of mycobacterial dissemination in mice.