scholarly journals Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Pazit Y. Cohen ◽  
Raphael Breuer ◽  
Philip Zisman ◽  
Shulamit B. Wallach-Dayan
Keyword(s):  
Mouse Lung ◽  
Genetic Changes ◽  
Abnormal Accumulation ◽  
Inflammatory Milieu ◽  
The Impact ◽  
Mouse Lungs ◽  
Two Populations ◽  
Deficient Mice ◽  
Chip Analysis

Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1−) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1+lymphocytes and Thy1−myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

scholarly journals Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

2016 ◽  
Vol 473 (17) ◽  
pp. 2671-2685 ◽  
Author(s):  
Genta Ito ◽  
Kristina Katsemonova ◽  
Francesca Tonelli ◽  
Pawel Lis ◽  
Marco A.S. Baptista ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Mouse Lung ◽  
Rab Gtpase ◽  
Leucine Rich Repeat ◽  
Serine Residue ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
The Impact ◽  
Effector Binding

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

scholarly journals Perturbations to lysyl oxidase expression broadly influence the transcriptome of lung fibroblasts

2017 ◽  
Vol 49 (8) ◽  
pp. 416-429 ◽  
Author(s):  
Ivana Mižíková ◽  
Francesco Palumbo ◽  
Tamás Tábi ◽  
Susanne Herold ◽  
István Vadász ◽  
...  
Keyword(s):  
Steady State ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Mouse Lung ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
Lysyl Oxidases ◽  
Primary Mouse ◽  
The Impact

Lysyl oxidases are credited with pathogenic roles in lung diseases, including cancer, fibrosis, pulmonary hypertension, congenital diaphragmatic hernia, and bronchopulmonary dysplasia (BPD). Lysyl oxidases facilitate the covalent intra- and intermolecular cross-linking of collagen and elastin fibers, thereby imparting tensile strength to the extracellular matrix (ECM). Alternative ECM-independent roles have recently been proposed for lysyl oxidases, including regulation of growth factor signaling, chromatin remodeling, and transcriptional regulation, all of which impact cell phenotype. We demonstrate here that three of the five lysyl oxidase family members, Lox, Loxl1, and Loxl2, are highly expressed in primary mouse lung fibroblasts compared with other constituent cell types of the lung. Microarray analyses revealed that small interfering RNA knockdown of Lox, Loxl1, and Loxl2 was associated with apparent changes in the expression of 134, 3,761, and 3,554 genes, respectively, in primary mouse lung fibroblasts. The impact of lysyl oxidase expression on steady-state Mmp3, Mmp9, Eln, Rarres1, Gdf10, Ifnb1, Csf2, and Cxcl9 mRNA levels was validated, which is interesting, since the corresponding gene products are relevant to lung development and BPD, where lysyl oxidases play a functional role. In vivo, the expression of these genes broadly correlated with Lox, Loxl1, and Loxl2 expression in a mouse model of BPD. Furthermore, β-aminopropionitrile (BAPN), a selective lysyl oxidase inhibitor, did not affect the steady-state mRNA levels of lysyl oxidase target genes, in vitro in lung fibroblasts or in vivo in BAPN-treated mice. This study is the first to report that lysyl oxidases broadly influence the cell transcriptome.

scholarly journals MÖNCH detector enables fast and low-dose free-propagation phase-contrast computed tomography ofin situmouse lungs

2018 ◽  
Vol 25 (2) ◽  
pp. 565-569 ◽  
Author(s):  
Christian Dullin ◽  
Jonas Albers ◽  
Giuliana Tromba ◽  
Marie Andrä ◽  
Marco Ramilli ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Lung Disease ◽  
Phase Contrast ◽  
Mouse Lung ◽  
The Novel ◽  
Entrance Dose ◽  
Hybrid Detector ◽  
Dose Levels ◽  
Mouse Lungs

Due to the complexity of the underlying pathomechanism,in vivomouse lung-disease models continue to be of great importance in preclinical respiratory research. Longitudinal studies following the cause of a disease or evaluating treatment efficacy are of particular interest but challenging due to the small size of the mouse lung and the fast breathing rate. Synchrotron-based in-line phase-contrast computed tomography imaging has been successfully applied in lung research in various applications, but mostly at dose levels that forbid longitudinalin vivostudies. Here, the novel charge-integrating hybrid detector MÖNCH is presented, which enables imaging of mouse lungs at a pixel size of 25 µm, in less than 10 s and with an entrance dose of about 70 mGy, which therefore will allow longitudinal lung disease studies to be performed in mouse models.

scholarly journals The Novel β-Lactam Enhancer Zidebactam Augments theIn VivoPharmacodynamic Activity of Cefepime in a Neutropenic Mouse LungAcinetobacter baumanniiInfection Model

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
S. S. Bhagwat ◽  
H. Periasamy ◽  
S. S. Takalkar ◽  
S. R. Palwe ◽  
H. N. Khande ◽  
...  
Keyword(s):  
Bactericidal Effect ◽  
Multidrug Resistant ◽  
Mouse Lung ◽  
Infection Model ◽  
Bactericidal Action ◽  
Content Type ◽  
Time Kill ◽  
The Impact

ABSTRACTWCK 5222 is a combination of cefepime and the high-affinity PBP2-binding β-lactam enhancer zidebactam. The cefepime-zidebactam combination is active against multidrug-resistant Gram-negative bacteria, including carbapenemase-expressingAcinetobacter baumannii. The mechanism of action of the combination involves concurrent multiple penicillin binding protein inhibition, leading to the enhanced bactericidal action of cefepime. The aim of the present study was to assess the impact of the zidebactam-mediated enhancedin vitrobactericidal action in modulating the percentage of the time that the free drug concentration remains above the MIC (percentfT>MIC) for cefepime required for thein vivokilling ofA. baumannii. Cefepime and cefepime-zidebactam MICs were comparable and ranged from 2 to 16 mg/liter for theA. baumanniistrains (n = 5) employed in the study. Time-kill studies revealed the improved killing of these strains by the cefepime-zidebactam combination compared to that by the constituents alone. Employing a neutropenic mouse lung infection model, exposure-response analyses for all theA. baumanniistrains showed that the cefepimefT>MIC required for 1-log10kill was 38.9%. In the presence of a noneffective dose of zidebactam, the cefepimefT>MIC requirement dropped significantly to 15.5%, but it still rendered a 1-log10kill effect. Thus, zidebactam mediated the improvement in cefepime’s bactericidal effect observed in time-kill studies, manifestedin vivothrough the lowering of cefepime’s pharmacodynamic requirement. This is a first-ever study demonstrating a β-lactam enhancer role of zidebactam that helps augment thein vivoactivity of cefepime by reducing the magnitude of its pharmacodynamically relevant exposures againstA. baumannii.

scholarly journals An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4915-4923 ◽  
Author(s):  
Akiko Shigeta ◽  
Masanori Matsumoto ◽  
Thomas F. Tedder ◽  
John B. Lowe ◽  
Masayuki Miyasaka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neutrophil Recruitment ◽  
Cremaster Muscle ◽  
Double Knockout ◽  
Independent Manner ◽  
Peripheral Blood Neutrophil ◽  
Selectin Ligand ◽  
The Impact ◽  
Deficient Mice

Abstract Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin–mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin–mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1–deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1–deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1–deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1–independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.

scholarly journals Retinaldehyde Dehydrogenase 1 Coordinates Hepatic Gluconeogenesis and Lipid Metabolism

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3089-3099 ◽  
Author(s):  
Florian W. Kiefer ◽  
Gabriela Orasanu ◽  
Shriram Nallamshetty ◽  
Jonathan D. Brown ◽  
Hong Wang ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Protein Kinase ◽  
Glucose Homeostasis ◽  
Retinoid Metabolism ◽  
Hepatic Glucose ◽  
Amp Activated Protein Kinase ◽  
The Impact ◽  
Deficient Mice

Recent data link vitamin A and its retinoid metabolites to the regulation of adipogenesis, insulin sensitivity, and glucose homeostasis. Retinoid metabolism is tightly controlled by an enzymatic network in which retinaldehyde dehydrogenases (Aldh1–3) are the rate-limiting enzymes that convert retinaldehyde to retinoic acid. Aldh1a1-deficient mice are protected from diet-induced obesity and hence diabetes. Here we investigated whether Aldh1a1 and the retinoid axis regulate hepatic glucose and lipid metabolism independent of adiposity. The impact of Aldh1a1 and the retinoid pathway on glucose homeostasis and lipid metabolism was analyzed in hepatocytes in vitro and in chow-fed, weight-matched Aldh1a1-deficient vs. wild-type (WT) mice in vivo. Aldh1a1-deficient mice displayed significantly decreased fasting glucose concentrations compared with WT controls as a result of attenuated hepatic glucose production. Expression of key gluconeogenic enzymes as well as the activity of Forkhead box O1 was decreased in Aldh1a1-deficient vs. WT livers. In vitro, retinoid or cAMP agonist stimulation markedly induced gluconeogenesis in WT but not Aldh1a1-deficient primary hepatocytes. Aldh1a1 deficiency increased AMP-activated protein kinase α activity, decreased expression of lipogenic targets of AMP-activated protein kinase α and significantly attenuated hepatic triacylglycerol synthesis. In metabolic cage studies, lean Aldh1a1-deficient mice manifested enhanced oxygen consumption and reduced respiratory quotient vs. WT controls, consistent with increased expression of fatty acid oxidation markers in skeletal muscle. Taken together, this work establishes a role for retinoid metabolism in glucose homeostasis in vivo and for Aldh1a1 as a novel determinant of gluconeogenesis and lipid metabolism independent of adiposity.

scholarly journals Loss of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis

2020 ◽  
Author(s):  
H Furkan Alkan ◽  
Paul W Vesely ◽  
Hubert Hackl ◽  
Johannes Foßelteder ◽  
Matthew G Vander Heiden ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pulmonary Metastasis ◽  
Mouse Lung ◽  
Intravenous Injections ◽  
Metastatic Capacity ◽  
Patient Prognosis ◽  
The Impact ◽  
Malate Aspartate Shuttle

AbstractBackgroundAspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of aspartate synthesis on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis.ResultsAGC1 expression is positively correlated with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells.ConclusionThis study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation do not always reflect the metastatic capacity in vivo.

Populations of NGF-dependent neurones differ in their requirement for BAX to undergo apoptosis in the absence of NGF/TrkA signalling in vivo

Development ◽  
2001 ◽  
Vol 128 (23) ◽  
pp. 4715-4728
Author(s):  
Gayle Middleton ◽  
Alun M. Davies
Keyword(s):  
Trigeminal Ganglion ◽  
Neuronal Death ◽  
Normal Development ◽  
Naturally Occurring ◽  
Ngf Receptor ◽  
Cervical Ganglion ◽  
Different Populations ◽  
Two Populations ◽  
Deficient Mice

Reports that apoptosis within populations of neurotrophin-dependent neurones is virtually eliminated in BAX-deficient mice and that BAX-deficient neurones survive indefinitely in culture without neurotrophins have led to the view that BAX is required for the death of neurotrophin-deprived neurones. To further examine this assertion in vivo, we have studied two populations of NGF-dependent neurones during the period of naturally occurring neuronal death in mice that lack BAX, NGF or the NGF receptor TrkA, alone and in combination. In the superior cervical ganglion (SCG), naturally occurring neuronal death and the massive loss of neurones that took place in the absence of NGF or TrkA were completely prevented by elimination of BAX. However, in the trigeminal ganglion, naturally occurring neuronal death was only partly abrogated by the elimination of BAX, and although the massive neuronal death that took place in this ganglion in the absence of NGF or TrkA was initially delayed in embryos lacking BAX, this subsequently occurred unabated. Accordingly, BAX-deficient neurones survived in defined without NGF whereas BAX-deficient trigeminal neurones died in the absence of NGF. These results indicate that whereas BAX is required for the death of SCG neurones during normal development and when these neurones are deprived of NGF/TrkA signalling in vivo, the death of trigeminal ganglion neurones occurs independently of BAX when they are deprived of NGF/TrkA signalling. We conclude that BAX is not universally required for neuronal death induced by neurotrophin deprivation, but that there are major differences for the requirement for BAX among different populations of NGF-dependent neurones.

scholarly journals Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets

2019 ◽  
Vol 217 (3) ◽  
Author(s):  
Maryam Ghaedi ◽  
Zi Yi Shen ◽  
Mona Orangi ◽  
Itziar Martinez-Gonzalez ◽  
Lisa Wei ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Retinoic Acid Receptor ◽  
Allergic Inflammation ◽  
Lymphoid Cells ◽  
Orphan Receptor ◽  
Mouse Lung ◽  
Mouse Lungs

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor–related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2− population different from conventional IL-18Rα−ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

scholarly journals Deficiency of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
H. Furkan Alkan ◽  
Paul W. Vesely ◽  
Hubert Hackl ◽  
Johannes Foßelteder ◽  
Daniel R. Schmidt ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pulmonary Metastasis ◽  
Mouse Lung ◽  
Intravenous Injections ◽  
Metastatic Capacity ◽  
Patient Prognosis ◽  
The Impact ◽  
Malate Aspartate Shuttle

Abstract Background Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. Results Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. Conclusion This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.

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