Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model

<p>Abstract</p> <p>Objective</p> <p>The phenomena manifested during inflammation require interplay between circulating effector cells, local resident cells, soluble mediators and genetic host factors to establish, develop and maintain itself. Of the molecues involed in the initiation and perpetuation of acute allergic inflammation in asthma, the involvement of effector cells in redox reactions for producing O₂^-(superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them.</p> <p>Materials and Methods and Treatment</p> <p>To clarify the role of gp91phox subunit of NADPH oxidase in the development and progression of an acute allergic asthma phenotype, we induced allergen dependent inflammation in a gp91<it>^phox</it>-/- single knockout and a gp91phox-/-MMP-12-/- double knockout mouse models.</p> <p>Results</p> <p>In the knockout mice, both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicate a regulatory role for NADPH oxidase in development of allergic asthma. While T cell mediated functions like Th2 cytokine secretion, and proliferation to OVA were upregulated synchronous with the overall robustness of the asthma phenotype, macrophage upregulation in functions such as proliferation, and mixed lymphocyte reaction indicate a regulatory role for gp91phox and an overall non-involvement or synergistic involvement of MMP12 in the response pathway (comparing data from gp91phox-/- and gp91phox-/-MMP-12-/- mice).</p

NADPH Oxidase has a Regulatory Role in Acute Allergic Asthma

Objective: For the establishment of inflammation, a constant interplay between different effector cells from circulation, local resident cells, soluble mediators and genetic host factors is required. Molecular mechanisms, initiating and perpetuating inflammation, in particular, the involvement of effector cells in redox reactions for producing O2- (superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them. Materials, Methods and Treatment: To clarify the role of NADPH oxidase in T cell-initiated, macrophage-associated allergic asthma, we induced allergen dependent inflammation in a gp91phox-/- mouse. Results: Both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicates a regulatory role for NADPH oxidase in development of allergic asthma. While T cell-mediated functions like Th2 cytokine secretion, and proliferation to OVA were up-regulated synchronous with the overall robustness of the asthma phenotype, macrophage up-regulation in functions such as proliferation, mixed lymphocyte reaction, and MCP-1 directed chemotaxis, but downregulation of respiratory burst response indicates a forking in their signaling pathways. gp91phox-/- MMP12 double knockout (DKO) mice show a similar phenotype as the gp91phox-/- showing the non-involvement or synergistic involvement of MMP12 in the response pathway. In mixed lymphocyte reaction using the Increased B7.1 but reduced B7.2 and MHC class II expression indicating alteration of co-stimulatory molecule expression critical for T cell activation on both gp91phox-/- and DKO mice may explain the mechanism by which gp91phox may regulate Th2 pathway in allergic asthma

Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model

Our aim was to differentiate human (h) embryonic stem (ES) cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI) and type II (AEII) cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF). A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB) differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.Omission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP) transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC(-/-) mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.This study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis

The global burden of cancer attributable to risk factors, 2010-19 : a systematic analysis for the Global Burden of Disease Study 2019

Background Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. Methods The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk-outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. Findings Globally, in 2019, the risk factors included in this analysis accounted for 4.45 million (95% uncertainty interval 4.01-4.94) deaths and 105 million (95.0-116) DALYs for both sexes combined, representing 44.4% (41.3-48.4) of all cancer deaths and 42.0% (39.1-45.6) of all DALYs. There were 2.88 million (2.60-3.18) risk-attributable cancer deaths in males (50.6% [47.8-54.1] of all male cancer deaths) and 1.58 million (1.36-1.84) risk-attributable cancer deaths in females (36.3% [32.5-41.3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20.4% (12.6-28.4) and DALYs by 16.8% (8.8-25.0), with the greatest percentage increase in metabolic risks (34.7% [27.9-42.8] and 33.3% [25.8-42.0]). Interpretation The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.Peer reviewe

Reversal of lung inflammation and fibrosis by intratracheal transplantation of differentiated hES cells.

<p>Transplant groups consisted of <b>a</b>, <b>e</b>, <b>i–n</b> saline-treated control Rag2γC^−/− mice (Saline group), <b>c</b>, <b>g</b>, <b>i–n</b> bleomycin-treated Rag2γC^−/− mice administered saline (Bleo/Saline group), and bleomycin-treated Rag2γC^−/− mice transplanted with 10⁵ H7 hES cells differentiated in SAGM in the either the <b>c</b>, <b>g</b>, <b>i–n</b> absence (Bleo/hES+SAGM group), or <b>d</b>, <b>h</b>, <b>i–n</b> presence of 5 µM ICG-001 (Bleo/hES+SAGM+ICG-001 group) were stained with either <b>a–d</b> Masson's trichrome or <b>e–h</b> Picro Sirius red staining for collagen. 60× magnification. <b>i</b> shows BAL fluid cell counts for macrophages (solid bars), lymphocytes (hatched bars), and neutrophils (open bars) and <b>j</b> total soluble collagen/lung measured by the Sircol™ assay. <b>k–n</b> show expression by qPCR in relative units of the following genes: <b>k</b> collagen genes 1α2, 3α1, and 6α1 [Col 1α2 (white bars), Col 3α1 (black bars), Col 6α1 (gray bars)], <b>l</b> [TGFβ₁ (white bars), TGFβ₂ (black bars), and TGFβ₃ (grey bars)], <b>m</b> FGF [FGF-1 (white bars) and FGF-2 (black bars)], and <b>n</b> VEGF [VEGF-A (white bars), VEGF-B (black bars), and VEGF-C (gray bars)]. <i>P</i><0.05 values compared to bleomycin-treated control group administered saline are shown. Three independent experiments were performed with <i>n</i> = 4 mice per study group in each experiment. <b>i</b> BAL fluid counts and <b>k–n</b> qPCR data were performed in duplicate, and <b>j</b> collagen levels were measured in triplicate.</p

Phenotypic analysis and ultrastructure of hES cells differentiated to lung epithelial cell-specific lineages.

<p>EBs derived from H7 hES cells were allowed to form outgrowths and differentiate in EB medium over 10 days and then cells cultured over 12 days in either <b>a</b>–<b>d</b>, <b>i</b>, <b>j</b> SAGM or <b>e</b>–<b>h</b>, <b>k</b> BEGM. Expression of intracellular marker proteins was quantitated by <b>a</b> FACS analysis of single cell suspensions and <b>e</b> IF microscopy with anti-AQP-5, SP-C, and CC-10 monoclonal antibodies used to identify AEI cells, AEII cells, and Clara cells respectively. The mean percentage positive of total cells is shown ± SEM. <b>b</b>, <b>f</b> qPCR of mRNA of AQP-5, SP-C, and CC-10 normalized to human GAPDH is expressed as relative units ± SEM. Symbols: -???-, AQP-5; -▪-, SP-C; -•-, CC-10. <b>a</b>, <b>b</b>, <b>e</b>, <b>f</b> The data are reported as mean ± SEM; <i>n</i> = 3 independent experiments with <b>a</b> flow data collected in triplicate and <b>b</b>, <b>f</b> qPCR and <b>e</b> IF data collected in duplicate. The asterisk indicates that all values were significant (<i>P</i><0.05) for <b>a</b>, <b>b</b> SP-C and <b>e</b>, <b>f</b> CC-10 for days 4–12 in culture when compared against day 0. The individual <i>P</i><0.05 values for days 4–12 in culture for the marker data points are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033165#pone.0033165.s006" target="_blank">Table S3</a></b>. <b>c</b>, <b>g</b> H7 hES cells differentiated into epithelial cells viewed at 10× magnification with <b>d</b> SP-C-FITC⁺ and <b>h</b> CC-10-FITC⁺ cells shown by IF microscopy. <b>i</b>, <b>j</b> As seen by transmission electron microscopy, representative AEII cells grown in SAGM at day 12 contain characteristic cytoplasmic lamellar bodies (<b>i</b>, arrows, scale bar = 1 µm and <b>j</b>, scale bar = 200 nm). <b>k</b> Clara cell grown in BEGM at day 12 by transmission electron microscopy exhibit apical microvilli on cell surface (arrows) and electron dense secretory vesicles (circle), scale bar = 0.5 µm.</p

Effect of ICG-001 on differentiation of hES cells in SAGM to AEI cells.

<p>Single cell suspensions of H7 hES cells differentiated in SAGM were incubated with 5 µM ICG-001 in culture medium for 12 h and assayed by FACS to identify percentage of AEII cells (SP-C⁺), AEI cells (AQP-5⁺), and Clara cells (CC-10⁺). Percentage of total cells in culture of cells positive for <b>a</b> intracellular and <b>b</b> surface expression of AEI cell, AEII cell, and Clara cell markers. Surface expression of <b>d</b> pluripotent markers for hematopoietic cells (c-kit⁺), lineage-negative (Lin⁻) cells, and Oct3/4⁺ pluripotent cells, and <b>e</b> growth factors (EGF⁺, VEGF⁺, IGFII⁺). The percentage of positive cells of total cells in culture is shown as mean ± SEM (<i>n</i> = three independent experiments with FACS analyses performed in triplicate). <i>P</i><0.05 values in ICG-001-treated group (open bars) vs. ICG-001-untreated group (solid bars) are shown. Representative FACS scattergrams with the percentage of cells gated in each quadrant shown for expression of <b>c</b> AQ-5 (UL, 0.03%; UR, 5.08%; LL, 4.92%; and LR, 89.97%) and <b>e</b>: Oct3/4 and VEGF (UL, 1.47%; UR, 0.53%; LL, 86.15%; and LR, 11.85%).</p