Dianne M. Walters

Antiviral Activity of Nrf2 in a Murine Model of Respiratory Syncytial Virus Disease

RATIONALE Respiratory syncytial virus (RSV) is the most frequent cause of significant lower respiratory illness in infants and young children, but its pathogenesis is not fully understood. The transcription factor Nrf2 protects lungs from oxidative injury and inflammation via antioxidant response element (ARE)-mediated gene induction. OBJECTIVES The current study was designed to determine the role of Nrf2-mediated cytoprotective mechanisms in murine airway RSV disease. METHODS Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice were intranasally instilled with RSV or vehicle. In a separate study, Nrf2(+/+) and Nrf2(-/-) mice were treated orally with sulforaphane (an Nrf2-ARE inducer) or phosphate-buffered saline before RSV infection. MEASUREMENTS AND MAIN RESULTS RSV-induced bronchopulmonary inflammation, epithelial injury, and mucus cell metaplasia as well as nasal epithelial injury were significantly greater in Nrf2(-/-) mice than in Nrf2(+/+) mice. Compared with Nrf2(+/+) mice, significantly attenuated viral clearance and IFN-gamma, body weight loss, heightened protein/lipid oxidation, and AP-1/NF-kappaB activity along with suppressed antioxidant induction was found in Nrf2(-/-) mice in response to RSV. Sulforaphane pretreatment significantly limited lung RSV replication and virus-induced inflammation in Nrf2(+/+) but not in Nrf2(-/-) mice. CONCLUSIONS The results of this study support an association of oxidant stress with RSV pathogenesis and a key role for the Nrf2-ARE pathway in host defense against RSV.

Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice

BackgroundMulti-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis.MethodsMWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA.ResultsMice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung.ConclusionsThese results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner

BackgroundElevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM) contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide (V2O5) is a component of PM derived from fuel combustion as well as a source of occupational exposure in humans. In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion) model of pulmonary neoplasia in mice.ResultsA/J, BALB/cJ (BALB), and C57BL/6J (B6) mice were treated either with the initiator 3-methylcholanthrene (MCA; 10 μg/g; i.p.) or corn oil followed by 5 weekly aspirations of V2O5 or PBS and pulmonary tumors were enumerated 20 weeks following MCA treatment. Susceptibility to V2O5-induced pulmonary inflammation was assessed in bronchoalveolar lavage fluid (BALF), and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 ± 0.9 tumors/mouse) and BALB (2.2 ± 0.36) mice significantly above that observed with MCA/PBS or V2O5 alone (P < 0.05). No tumors were observed in the B6 mice in any of the experimental groups. Mice sensitive to tumor promotion by V2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6). Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFκB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue.ConclusionsIn this study we demonstrate that V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred strains of mice. Further, we identified a positive relationship between tumor promotion and susceptibility to V2O5-induced pulmonary inflammation. These findings suggest that repeated exposures to V2O5 containing particles may augment lung carcinogenesis in susceptible individuals through oxidative stress mediated pathways.

Susceptibility of Signal Transducer and Activator of Transcription-1-Deficient Mice to Pulmonary Fibrogenesis

The signal transducer and activator of transcription (Stat)-1 mediates growth arrest and apoptosis. We postulated that lung fibrosis characterized by excessive proliferation of lung fibroblasts would be enhanced in Stat1-deficient (Stat1-/-) mice. Two weeks after bleomycin aspiration (3 U/kg), Stat1-/- mice exhibited a more severe fibroproliferative response and significantly elevated total lung collagen compared to wild-type mice. Growth factors [epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)] enhanced [3H]thymidine uptake in lung fibroblasts isolated from Stat1-/- mice compared to wild-type mice. Interferon (IFN)-gamma, which signals growth arrest via Stat1, inhibited EGF- or PDGF-stimulated mitogenesis in wild-type fibroblasts but enhanced [3H]thymidine uptake in Stat1-/- fibroblasts. Moreover, IFN-gamma treatment in the absence of growth factors induced a concentration-dependent increase in [3H]thymidine uptake in Stat1-/- but not wild-type fibroblasts. Mitogen-activated protein kinase (ERK-1/2) phosphorylation in response to PDGF or EGF did not differ among Stat1-/- and wild-type fibroblasts. However, Stat3 phosphorylation induced by PDGF, EGF, or IFN-gamma increased twofold in Stat1-/- fibroblasts compared to wild-type fibroblasts. Our findings indicate that Stat1-/- mice are more susceptible to bleomycin-induced lung fibrosis than wild-type mice due to 1) enhanced fibroblast proliferation in response to growth factors (EGF and PDGF), 2) stimulation of fibroblast growth by a Stat1-independent IFN-gamma signaling pathway, and 3) increased activation of Stat3.

Mast cells contribute to altered vascular reactivity and ischemia-reperfusion injury following cerium oxide nanoparticle instillation

Abstract Cerium oxide (CeO2) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO2 exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-KitW-sh mast cell deficient mice following CeO2 nanoparticle instillation. C57BL/6 mice instilled with CeO2 exhibited mild pulmonary inflammation. However, B6.Cg-KitW-sh mice did not display a similar degree of inflammation following CeO2 instillation. Moreover, C57BL/6 mice instilled with CeO2 exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-KitW-sh mice. In vitro CeO2 exposure resulted in increased production of PGD2, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO2 nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

Mouse models of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is a component of many interstitial lung diseases, including idiopathic pulmonary fibrosis, a chronic, progressive disease for which there is currently no effective therapy. Bleomycin has been widely used in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and for evaluation of potential therapies. Bleomycin induces DNA strand breaks, resulting in pulmonary inflammation, injury, and subsequent interstitial fibrosis. This unit describes methods for delivering bleomycin, either directly into the lung or systemically, to create models of pulmonary fibrosis in rodents. Also described is a rapid and easy procedure for measuring lung collagen content to quantify the severity of fibrosis. Curr. Protoc. Pharmacol. 40:5.46.1‐5.46.17.

Vanadium-induced STAT-1 activation in lung myofibroblasts requires H2O2 and p38 map kinase

Vanadium compounds present in air pollution particulate matter activate signal transduction pathways in pulmonary cell types leading to pathological outcomes including aberrant cell proliferation, apoptosis, and cytokine expression. Vanadium has been proposed to activate transcription factors via the generation of hydrogen peroxide (H2O2). We investigated the mechanisms through which vanadium pentoxide (V2O5), the major form of vanadium released from the industrial burning of fuel oil, activated the signal transducer and activator of transcription (STAT)-1. V2O5-induced STAT-1 activation was blocked by catalase and N-acetyl-L-cysteine (NAC), suggesting vanadium-induced generation of H2O2. Surprisingly, however, V2O5 did not increase H2O2 levels released by rat lung myofibroblasts into cell culture supernatants. Instead, these quiescent myofibroblasts spontaneously released micromolar concentrations of H2O2, and the addition of V2O5 reduced H2O2 levels in cell culture supernatants within minutes. V2O5 suppressed H2O2 for as long as 24 h. Differences in the temporal activation of STAT-1 and p38 MAPK were observed following V2O5 or H2O2 treatment, and STAT-1 activation by V2O5 or H2O2 was attenuated by an inhibitor of the EGF receptor tyrosine kinase (AG1478) or p38 MAPK (SB203580). The phosphorylation of p38 MAPK by V2O5 was inhibited by NAC and catalase, yet the EGF receptor inhibitor AG1478 had no effect on V2O5-induced p38 MAPK activation. Collectively, our findings support the novel hypothesis that H2O2 spontaneously generated by myofibroblasts fuels vanadium-induced activation of STAT-1. Moreover, p38 MAPK and EGF receptor activation are required for V2O5-induced STAT-1 activation.

Paternal long-term exercise programs offspring for low energy expenditure and increased risk for obesity in mice

Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin‐resistant phenotype. Although diet‐induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long‐term voluntary wheel‐running in C57BL/6J male mice on their offsprings predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel‐running for 12 wk produced offspring that were more susceptible to the adverse effects of a high‐fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long‐term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro‐RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA‐induced modification of sperm.—Murashov, A. K., Pak, E. S., Koury, M., Ajmera, A., Jeyakumar, M., Parker, M., Williams, O., Ding, J., Walters, D., Neufer, P. D. Paternal long‐term exercise programs offspring for low energy expenditure and increased risk for obesity in mice. FASEB J. 30, 775–784 (2016). www.fasebj.org

Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5)

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose‐dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5‐induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5‐induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.—Walters, D. M., White, K. M., Patel, U., Davis, M. J., Veluci‐Marlow, R. M., Bhupanapadu Sunkesula, S. R., Bonner, J. C., Martin, J. R., Gladwell, W., Kleeberger, S. R. Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5). FASEB J. 28, 1098–1112 (2014). www.fasebj.org