Gene expression profiles of Calu-3 lung epithelial cells after neutrophil transmigration
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE31697 | GEO | 2011/08/28
SECONDARY ACCESSION(S): PRJNA145623
REPOSITORIES: GEO
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Project description:Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. Herein, we used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiologic basolateral-to-apical direction to a gradient of fMLP. Control wells were treated with fMLP alone. Indicated wells were pretreated with an anti-CD47 blocking antibody, which exacerbates epithelial injury. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifest by restoration of transepithelial resistance (TER) and re-epithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. This pathway represents a potential therapeutic target to accelerate physiologic recovery in inflammatory lung diseases. Total of 20 samples: Calu-3 cells that were untreated, treated with fMLP, treated with fMLP and neutrophil transmigration, and treated with anti-CD47 antibodies, fMLP, and neutrophil transmigration; cDNA was pooled from n=3 experiments; No biological replicates
2011-08-27 | E-GEOD-31697 | biostudies-arrayexpress
Project description:Neutrophil accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. Based on recent evidence that mucosal metabolic changes influence disease outcomes, we hypothesized that transmigrating neutrophils influence the transcriptional profile of intestinal epithelia. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by neutrophil-epithelial crosstalk. Real-time O2 sensing indicated that transmigrating neutrophils rapidly deplete microenvironmental O2 sufficient enough to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the relative contribution of neutrophils and the respiratory burst to M-bM-^@M-^\inflammatory hypoxiaM-bM-^@M-^] in vivo. Gp91phox-null mice, which mirror human chronic granulomatous disease, developed accentuated colitis compared to control with exaggerated neutrophil infiltration and diminished inflammatory hypoxia. In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils modulates the host response to inflammation. Likewise, the respiratory burst contributes fundamentally to localized O2 depletion, resultant microenvironmental hypoxia and effective inflammatory resolution. Two models were employed, direct and indirect. The M-bM-^@M-^\DirectM-bM-^@M-^] migration model entailed establishing a chemotactic gradient (using fMLP) across monolayers of T84 intestinal epithelial cells grown on the underside of permeable supports (3um pore). Neutrophils (PMN) were induced to migrate in the physiologically relevant the physiologically relevant basolateral-to-apical direction. Following migration, T84s were rested in complete media and 2hrs later harvested for RNA isolation. In the Indirect model, PMN were applied to T84s as with the direct model. After migration, conditioned supernatants were collected, cells pelleted and supernatants filtered through 0.2um pore. Conditioned supernatants were transferred to naive T84 monolayers for 2hrs, followed by RNA harvest. Each model was exposed to neutrophils (PMN) or not. All monolayers contained chemotactic peptide fMLP on the apical side. Total of 12 samples, 4 conditions in triplicate: Direct migration without neutrophils (T84 +fMLP -PMN), Direct migration with neutrophils (T84 +fMLP +PMN), Indirect migration without neutrophils (T84 +fMLP -PMN), Indirect migration with neutrophils (T84 +fMLP +PMN)
2012-10-31 | E-GEOD-39681 | biostudies-arrayexpress
Project description:Wnt/β-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/β-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/β-catenin responsive progenitor cells and the potential impact of Wnt/β-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema, are poorly understood. Here, we used a TCF/Lef:H2B/GFP reporter mice to investigate the role of Wnt/β-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/β-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. To further characterize the lung epithelial populations with different Wnt activities, we perform microarray analysis using freshly isolated Wnthigh/low/negative lung epithelial cells to study their transcriptome, specially the enriched genes and signaling pathways in the Wnt low population related epithelial stem cell functions.
2020-06-12 | GSE150957 | GEO
Project description:This study investigates the role of regulatory T cell–derived Areg in driving epithelial repair following influenza infection. We use RNA sequencing to probe the gene expression changes within distinct mesenchymal cell subsets of the lung upon stimulation with EGF (50ng/mL), AREG (200ng/mL), or mock. n = 2 wells / group.
2022-12-14 | GSE169125 | GEO
Project description:Postnatal tissue quiescence is generally thought to be a default state in the absence of a proliferative stimulus such as injury. We now demonstrate that in the lung, quiescence in the adult is an actively maintained state and is regulated by paracrine hedgehog signaling. Epithelial-specific deletion of Sonic Hedgehog during normal homeostasis results in a proliferative expansion of the adjacent lung mesenchyme. Injury to the lung epithelium results in decreased hedgehog activation, accompanied by proliferative expansion of the adjacent mesenchyme. Moreover, reconstitution of Hedgehog signaling during epithelial injury attenuated the proliferative expansion of the adjacent mesenchyme. Hedgehog signaling maintains lung quiescence by attenuating PDGF signaling through blocking post-translational processing of PDGF receptor α/β into the mature isoforms. These results indicate that in postnatal tissues, epithelial cells can actively maintain mesenchymal quiescence via paracrine hedgehog activation, and that imbalances in this pathway could lead to aberrant mesenchymal expansion and postnatal disease. Fibroblasts were isolated from mouse lungs and grown in culture in triplicate wells. Samples were treated with vehicle or purmorphamine 5um for 24 hours and RNA was isolated for microarray.
2016-07-03 | E-GEOD-61026 | biostudies-arrayexpress
Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in unstimulated and fMLP-stimulated neutrophils derived from mouse bone marrow, we performed micorarray analysis of gene expression in unstimulated wild type (N=3), unstimulated lcn2 knockout (N=3), fMLP-stimulated wild type (N=2) and fMLP-stimulated lcn2 knockout (N=2) neutrophils. Upon stimulation, neutrophils were treated by fMLP at 10 micromolar for 20 minutes at 37 centigrade.
2013-01-31 | E-GEOD-43889 | biostudies-arrayexpress
Project description:Idiopathic Pulmonary Fibrosis (IPF) is a progressive and often fatal chronic respiratory disease thought to result from repetitive injury and failed repair of the lung alveoli, and recent studies have identified a number of disease-emergent intermediate/transitional cell states in the IPF lung supporting this concept. In this study, we found that persistent activation of hypoxia-inducible factor (HIF)-signaling in airway-derived, repair-associated cell types/states is a hallmark of dysfunctional epithelial repair in the IPF lung epithelium and experimental models of recurrent lung epithelial injury. Disrupting Hif-signaling attenuated experimental lung fibrosis, reduced mucous-secretory cell polarization, and promoted functional alveolar regeneration following repetitive injury. Mouse and human organoid studies demonstrated that small-molecule-based HIF2 inhibition promoted alveolar epithelial cell proliferation and maturation while preventing the emergence of maladaptive intermediate/transitional states analogous to those in IPF. Together, these studies indicate that targeted HIF2-inhibition represents a novel and effective therapeutic strategy to promote functional lung regeneration, and could be readily translated into human studies of IPF and other chronic interstitial lung diseases with disease modifying effect.
2023-09-18 | GSE243252 | GEO
Project description:Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair. Bronchiolar Clara cells undergo phenotypic changes during development and in disease. These changes are poorly described due to a paucity of molecular markers. We used chemical and transgenic approaches to ablate Clara cells, allowing identification of their unique gene expression profile. Flavin monooxygenase 3 (Fmo3), paraoxonase 1 (Pon1), aldehyde oxidase 3 (Aox3), and claudin 10 (Cldn10) were identified as novel Clara cell markers. New and existing Clara cell marker genes were categorized into three classes based on their unique developmental expression pattern. Cldn10 was uniformly expressed in the epithelium at Embryonic Day (E)14.5 and became restricted to secretory cells at E18.5. This transition was defined by induction of Clara Cell Secretory Protein (CCSP). Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Pon1, Aox3, and Cyp2f2 between late embryonic and postnatal periods. Messenger RNA abundance of all categories of genes was dramatically decreased after naphthalene-induced airway injury, and displayed a sequence of temporal induction during repair that suggested sequential secretory cell maturation. We have defined a broader repertoire of Clara cell-specific genes that allows staging of epithelial maturation during development and repair. 32 samples were isolated and used to screen UniSet Mouse 20K I Bioarrays (total lung RNA Mus musculus). Control RNA was isolated from uninjured mice (N = 4 mice), and RNA was also isolated from mice exposed to naphthalene and recovered for 1, 2, 3, and 6 days (N = 4 mice per timepoint). Additionally, transgenic mice expressing HSV-tk under control of the mouse CCSP promoter were exposed to Ganciclovir (GCV) and recovered for 3, 6, and 9 days (N = 4 mice per timepoint).
2009-08-18 | E-GEOD-17693 | biostudies-arrayexpress
Project description:Human volunteers were exposed to endotoxin by bronchoscopic instillation, airspace and circulating neutrophils were isolated 16 hours later and compared to circulating neutrophils obtained prior to endotoxin exposure, and to circulating neutrophils exposed to endotoxin in vitro. Keywords = pulmonary inflammantion Keywords = acute lung injury Keywords = acute respiratory distress syndrome Keywords: parallel sample
2006-01-01 | GSE2322 | GEO
Project description:Paraquat is a toxic herbicide that can cause severe lung injury, leading to alveolar epithelial cell death, subsequent lung fibrosis and respiratory failure. Understanding the repair process following paraquat injury is critical for developing effective therapeutic strategies to treat paraquat-poisoned patients. However, the lung repair program in paraquat-injured lungs is currently unknown. In this study, we analyzed lung parenchyma samples from an 18-year-old female patient who had ingested paraquat and subsequently underwent a double-lung transplantation on the 34th day after poisoning. By the combination of single cell RNA sequencing analysis and immunostaining analysis, we reveal that the airway stem/progenitor cells and AT2 cells in the paraquat-injured lung exhibit a significantly reduced capacity to regenerate alveolar epithelium epithelial in the paraquat-injured lung. The hypoxic microenvironment may contribute to this aberrant repair program by activating the NOTCH signaling pathway, ultimately leading to an irreversible loss of gas-exchange units and an exacerbated lung tissue damage.
2023-09-28 | GSE231647 | GEO