Alveolar Macrophage Chemokine Secretion Mediates Neutrophilic Lung Injury in Nox2-Deficient Mice.

Acute lung injury (ALI), developing as a component of the systemic inflammatory response syndrome (SIRS), leads to significant morbidity and mortality. Reactive oxygen species (ROS), produced in part by the neutrophil NADPH oxidase 2 (Nox2), have been implicated in the pathogenesis of ALI. Previous studies in our laboratory demonstrated the development of pulmonary inflammation in Nox2-deficient (gp91phox-/y) mice that was absent in WT mice in a murine model of SIRS. Given this finding, we hypothesized that Nox2 in a resident cell in the lung, specifically the alveolar macrophage, has an essential anti-inflammatory role. Using a murine model of SIRS, we examined whole-lung digests and bronchoalveolar lavage fluid (BALf) from WT and gp91phox-/y mice. Both genotypes demonstrated neutrophil sequestration in the lung during SIRS, but neutrophil migration into the alveolar space was only present in the gp91phox-/y mice. Macrophage inflammatory protein (MIP)-1α gene expression and protein secretion were higher in whole-lung digest from uninjected gp91phox-/y mice compared to the WT mice. Gene expression of MIP-1α, MCP-1, and MIP-2 was upregulated in alveolar macrophages obtained from gp91phox-/y mice at baseline compared with WT mice. Further, ex vivo analysis of alveolar macrophages, but not bone marrow-derived macrophages or peritoneal macrophages, demonstrated higher gene expression of MIP-1α and MIP-2. Moreover, isolated lung polymorphonuclear neutrophils migrate to BALf obtained from gp91phox-/y mice, further providing evidence of a cell-specific anti-inflammatory role for Nox2 in alveolar macrophages. We speculate that Nox2 represses the development of inflammatory lung injury by modulating chemokine expression by the alveolar macrophage.

Ex Vivo-expanded Natural Killer Cells Derived From Long-term Cryopreserved Cord Blood are Cytotoxic Against Primary Breast Cancer Cells.

With over 600,000 units of umbilical cord blood (CB) stored on a global scale, it is important to elucidate the therapeutic abilities of this cryopreserved reservoir. In the advancing field of natural killer (NK) cell cancer immunotherapy, CB has proven to be a promising and noninvasive source of therapeutic NK cells. Although studies have proven the clinical efficacy of using long-term cryopreserved CB in the context of hematopoietic stem cell transplantations, little is known about its use for the ex vivo expansion of effector immune cells. Therefore, our group sought to derive ex vivo-expanded NK cells from long-term cryopreserved CB, using an artificial antigen presenting cell-mediated expansion technique. We compared the expansion potential and antitumor effector function of CB-derived NK (CB-NK) cells expanded from fresh (n=4), short-term cryopreserved (<1-year old, n=5), and long-term cryopreserved (1-10-year old, n=5) CB. Here, we demonstrated it is possible to obtain an exponential amount of expanded CB-NK cells from long-term cryopreserved CB. Ex vivo-expanded CB-NK cells had an increased surface expression of activating markers and showed potent antitumor function by producing robust levels of proinflammatory cytokines, interferon-γ, and tumor necrosis factor-α. Moreover, expanded CB-NK cells (n=3-5) demonstrated cytotoxicity towards primary breast cancer cells (n=2) derived from a triple-negative breast cancer and an estrogen receptor-positive/progesterone receptor-positive breast cancer patient. Long-term cryopreservation had no effect on the expansion potential or effector function of expanded CB-NK cells. Therefore, we propose that long-term cryopreserved CB remains clinically useful for the ex vivo expansion of therapeutic NK cells.

The acetate/ACSS2 switch regulates HIF-2 stress signaling in the tumor cell microenvironment.

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals.

The acetylase/deacetylase couple CREB-binding protein/Sirtuin 1 controls hypoxia-inducible factor 2 signaling.

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors. HIF-1α plays a prominent role in hypoxic gene induction. HIF-2α target genes are more restricted but include erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals. We previously reported that HIF-2α is acetylated during hypoxia but is rapidly deacetylated by the stress-responsive deacetylase Sirtuin 1. We now demonstrate that the lysine acetyltransferases cAMP-response element-binding protein-binding protein (CBP) and p300 are required for efficient Epo induction during hypoxia. However, despite close structural similarity, the roles of CBP and p300 differ in HIF signaling. CBP acetylates HIF-2α, is a major coactivator for HIF-2-mediated Epo induction, and is required for Sirt1 augmentation of HIF-2 signaling during hypoxia in Hep3B cells. In comparison, p300 is a major contributor for HIF-1 signaling as indicated by induction of Pgk1. Whereas CBP can bind with HIF-2α independent of the HIF-2α C-terminal activation domain via enzyme/substrate interactions, p300 only complexes with HIF-2α through the C-terminal activation domain. Maximal CBP/HIF-2 signaling requires intact CBP acetyltransferase activity in both Hep3B cells as well as in mice.

Hypoxia increases sirtuin 1 expression in a hypoxia-inducible factor-dependent manner.

Hypoxia-inducible factors (HIFs) are stress-responsive transcriptional regulators of cellular and physiological processes involved in oxygen metabolism. Although much is understood about the molecular machinery that confers HIF responsiveness to oxygen, far less is known about HIF isoform-specific mechanisms of regulation, despite the fact that HIF-1 and HIF-2 exhibit distinct biological roles. We recently determined that the stress-responsive genetic regulator sirtuin 1 (Sirt1) selectively augments HIF-2 signaling during hypoxia. However, the mechanism by which Sirt1 maintains activity during hypoxia is unknown. In this report, we demonstrate that Sirt1 gene expression increases in a HIF-dependent manner during hypoxia in Hep3B and in HT1080 cells. Impairment of HIF signaling affects Sirt1 deacetylase activity as decreased HIF-1 signaling results in the appearance of acetylated HIF-2α, which is detected without pharmacological inhibition of Sirt1. We also find that Sirt1 augments HIF-2 mediated, but not HIF-1 mediated, transcriptional activation of the isolated Sirt1 promoter. These data in summary reveal a bidirectional link of HIF and Sirt1 signaling during hypoxia.

Regulation of hypoxia-inducible factor 2alpha signaling by the stress-responsive deacetylase sirtuin 1.

To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2alpha) during hypoxia. The effect of Sirt1 on HIF-2alpha required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2alpha that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2alpha target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.

Postpneumonectomy lung expansion elicits hypoxia-inducible factor-1alpha signaling.

We (42) previously reported differential regulation of hypoxia-inducible factors (HIF-1alpha, -2alpha, and -3alpha) mRNA in canine lungs during normal maturation and postpneumonectomy (PNX) compensatory growth in the absence of overt hypoxia. To test the hypothesis that lung expansion activates HIF signaling, we replaced the right lung of six adult foxhounds with inflated custom-shaped silicone prosthesis to keep the mediastinum in the midline and minimize lateral expansion of the remaining lung. After 3 wk of recovery and stabilization of perfusion, the prosthesis was acutely deflated in three animals, causing the remaining lung to expand by 114%. In three other animals, the prosthesis remained inflated. Three days following deflation, we observed significant elevation in the mRNA and nuclear protein levels of HIF-1alpha ( approximately 60%) as well as activation of its transcriptional regulator, the serine/threonine protein kinase B (phospho-Akt-to-total Akt ratio, 124%), and the mRNA and protein levels of its downstream targets, erythropoietin receptor (71-183%) as well as VEGF (33-58%) compared with the pre-PNX control lung from the same animal. The mRNA of HIF-2alpha, HIF-3alpha, and VEGF receptors did not change with acute deflation. We conclude that in vivo lung expansion by post-PNX deflation of space-occupying prosthesis elicits coordinated activation of HIF-1alpha signaling in adult lungs. This pathway could play an important role in mediating lung growth and remodeling during maturation and post-PNX compensation.

Splenectomy impairs diffusive oxygen transport in the lung of dogs.

The spleen acts as an erythrocyte reservoir in highly aerobic species such as the dog and horse. Sympathetic-mediated splenic contraction during exercise reversibly enhances convective O2 transport by increasing hematocrit, blood volume, and O2-carrying capacity. Based on theoretical interactions between erythrocytes and capillary membrane (Hsia CCW, Johnson RL Jr, and Shah D. J Appl Physiol 86: 1460-1467, 1999) and experimental findings in horses of a postsplenectomy reduction in peripheral O2-diffusing capacity (Wagner PD, Erickson BK, Kubo K, Hiraga A, Kai M, Yamaya Y, Richardson R, and Seaman J. Equine Vet J 18, Suppl: 82-89, 1995), we hypothesized that splenic contraction also augments diffusive O2 transport in the lung. Therefore, we have measured lung diffusing capacity (DL(CO)) and its components during exercise by a rebreathing technique in six adult foxhounds before and after splenectomy. Splenectomy eliminated exercise-induced polycythemia, associated with a 30% reduction in maximal O2 uptake. At any given pulmonary blood flow, DL(CO) was significantly lower after splenectomy owing to a lower membrane diffusing capacity, whereas pulmonary capillary blood volume changed variably; microvascular recruitment, indicated by the slope of the increase in DL(CO) with respect to pulmonary blood flow, was also reduced. We conclude that splenic contraction enhances both convective and diffusive O2 transport and provides another compensatory mechanism for maintaining alveolar O2 transport in the presence of restrictive lung disease or ambient hypoxia.

Retinoic acid-induced alveolar cellular growth does not improve function after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) treatment enhances lung function during compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 3 mo, transpulmonary pressure (TPP)-lung volume relationship, diffusing capacities for carbon monoxide and nitric oxide, cardiac output, and septal volume (V(tiss-RB)) were measured under anesthesia by a rebreathing technique at two lung volumes. Lung air and tissue volumes (V(air-CT) and V(tiss-CT)) were also measured from high-resolution computerized tomographic (CT) scans at a constant TPP. In RA-treated dogs compared with controls, TPP-lung volume relationships were similar. Diffusing capacities for carbon monoxide and nitric oxide were significantly impaired at a lower lung volume but similar at a high lung volume. Whereas V(tiss-RB) was significantly lower at both lung volumes in RA-treated animals, V(air-CT) and V(tiss-CT) were not different between groups; results suggest uneven distribution of ventilation consistent with distortion of alveolar geometry and/or altered small airway function induced by RA. We conclude that RA does not improve resting pulmonary function during the early months after R-PNX despite histological evidence of its action in enhancing alveolar cellular growth in the remaining lung.