Corrine R. Kliment

Neutrophil elastase–mediated degradation of IRS-1 accelerates lung tumor growth

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness. To determine the role of neutrophil elastase (encoded by Elane) on tumor progression, we used the loxP-Stop-loxP K-rasG12D (LSL–K-ras) model of mouse lung adenocarcinoma to generate LSL–K-ras-Elane−/− mice. Tumor burden was markedly reduced in LSL–K-ras-Elane−/− mice at all time points after induction of mutant K-ras expression. Kaplan-Meier survival analysis showed that whereas all LSL–K-ras-Elane+/+ mice died, none of the mice lacking neutrophil elastase died. Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells, where it degraded insulin receptor substrate-1 (IRS-1). Immunoprecipitation studies showed that, as neutrophil elastase degraded IRS-1, there was increased interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between neutrophil elastase and IRS-1 in LSL–K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS-1 as a key regulator of PI3K within malignant cells. Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive fibrosis of the alveolar interstitium. The pathogenesis is thought to involve abnormal reepithelialization and dysregulated remodeling of the extracellular matrix after alveolar injury. There is growing evidence through human and animal studies that oxidative stress plays a role in this dysregulation. Markers of oxidative stress have been identified in the lungs of IPF patients and aberrant antioxidant activity exacerbates pulmonary fibrosis in animal models. In addition, the extracellular matrix is a critical component in regulating cellular homeostasis and appropriate wound healing. Recent investigations support that the matrix is a target of oxidative stress in the lung and IPF. Extracellular matrix degradation products, produced by reactive oxygen species, may promote fibrogenesis by influencing epithelial, mesenchymal, and inflammatory cell activity. The impact of the interactions of oxidative stress and the matrix of the lung remains unclear and may prove to be an important target for new therapies in IPF. Utilizing oxidative enzymes, antioxidants, or the matrix as therapeutic targets to control oxidative stress in IPF will continue be an area of active research and innovative discoveries in the coming years.

Oxidative Stress Alters Syndecan-1 Distribution in Lungs with Pulmonary Fibrosis *□

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by severe, progressive fibrosis. Roles for inflammation and oxidative stress have recently been demonstrated, but despite advances in understanding the pathogenesis, there are still no effective therapies for IPF. This study investigates how extracellular superoxide dismutase (EC-SOD), a syndecan-binding antioxidant enzyme, inhibits inflammation and lung fibrosis. We hypothesize that EC-SOD protects the lung from oxidant damage by preventing syndecan fragmentation/shedding. Wild-type or EC-SOD-null mice were exposed to an intratracheal instillation of asbestos or bleomycin. Western blot was used to detect syndecans in the bronchoalveolar lavage fluid and lung. Human lung samples (normal and IPF) were also analyzed. Immunohistochemistry for syndecan-1 and EC-SOD was performed on human and mouse lungs. In vitro, alveolar epithelial cells were exposed to oxidative stress and EC-SOD. Cell supernatants were analyzed for shed syndecan-1 by Western blot. Syndecan-1 ectodomain was assessed in wound healing and neutrophil chemotaxis. Increases in human syndecan-1 are detected in lung homogenates and lavage fluid of IPF lungs. Syndecan-1 is also significantly elevated in the lavage fluid of EC-SOD-null mice after asbestos and bleomycin exposure. On IHC, syndecan-1 staining increases within fibrotic areas of human and mouse lungs. In vitro, EC-SOD inhibits oxidant-induced loss of syndecan-1 from A549 cells. Shed and exogenous syndecan-1 ectodomain induce neutrophil chemotaxis, inhibit alveolar epithelial wound healing, and promote fibrogenesis. Oxidative shedding of syndecan-1 is an underlying cause of neutrophil chemotaxis and aberrant wound healing that may contribute to pulmonary fibrosis.

CD4+ T cell–independent DNA vaccination against opportunistic infections

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Extracellular superoxide dismutase regulates cardiac function and fibrosis.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant that protects the heart from ischemia and the lung from inflammation and fibrosis. The role of cardiac EC-SOD under normal conditions and injury remains unclear. Cardiac toxicity, a common side effect of doxorubicin, involves oxidative stress. We hypothesize that EC-SOD is critical for normal cardiac function and protects the heart from oxidant-induced fibrosis and loss of function. C57BL/6 and EC-SOD-null mice were treated with doxorubicin, 15 mg/kg (i.p.). After 15 days, echocardiography was used to assess cardiac function. Left ventricle (LV) tissue was used to assess fibrosis and inflammation by staining, Western blot, and hydroxyproline analysis. At baseline, EC-SOD-null mice have LV wall thinning and increases in LV end diastolic dimensions compared to wild-type mice but have normal cardiac function. After doxorubicin, EC-SOD-null mice have decreases in fractional shortening not apparent in WT mice. Lack of EC-SOD also leads to increases in myocardial apoptosis and significantly more LV fibrosis and inflammatory cell infiltration. Administration of the metalloporphyrin AEOL 10150 abrogates the loss of cardiac function, and potentially fibrosis, associated with doxorubicin treatment in both wild-type and EC-SOD KO mice. EC-SOD is critical for normal cardiac morphology and protects the heart from oxidant-induced fibrosis, apoptosis, and loss of function. The antioxidant metalloporphyrin AEOL 10150 effectively protects cardiac function from doxorubicin-induced oxidative stress in vivo. These findings identify targets for the use of antioxidant agents in oxidant-induced cardiac fibrosis.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE−/− mice are protected from asbestos‐induced lung fibrosis. NE−/− mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF‐β‐independent fashion. Treatment of asbestos‐recipient mice with ONO‐5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE‐inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Experimental Models of Asbestos-Related Diseases

Much of our understanding of the mechanisms by which asbestos injures the lung has been derived from experimental animal studies. Such studies have confirmed the fibrogenic and carcinogenic properties of asbestos fibers that have been surmised from human observations and have provided insights into the ways in which asbestos fibers interact with biological systems. Models commonly used to study asbestos-induced disease involve inhalation exposure to asbestos, intratracheal instillation, and in vitro studies of various cellular systems. Each of these techniques has particular advantages and disadvantages.

Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding.

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.

Corrigendum. CD4+ T cell-independent DNA vaccination against opportunistic infections.

Original citation: J Clin Invest. 2005;115(12):3536–3544. doi:10.1172/JCI26306. Citation for this corrigendum: J Clin Invest. 2015;125(3):1364. doi:10.1172/JCI81228. Karen A. Norris has requested to be removed from the author list, and the corresponding author has agreed. The revised author list is shown above.

Erratum: CD4+ T cell'independent DNA vaccination against opportunistic infections (Journal of Clinical Investigation (2005) 115: 12 (3536-3544) DOI: 10.1172/JCI26306)

Original citation: J Clin Invest. 2005;115(12):3536–3544. doi:10.1172/JCI26306. Citation for this corrigendum: J Clin Invest. 2015;125(3):1364. doi:10.1172/JCI81228. Karen A. Norris has requested to be removed from the author list, and the corresponding author has agreed. The revised author list is shown above.