Dissecting the Niche for Alveolar Type II Cells With Alveolar Organoids.
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ABSTRACT:
SUBMITTER: Liao D
PROVIDER: S-EPMC7287157 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Dissecting the Niche for Alveolar Type II Cells With Alveolar Organoids.
Frontiers in cell and developmental biology 20200604
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Project description:Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1(-/-) mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1(-/-) mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1(-/-) mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFkappaB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn(-/-) mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.
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Project description:Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin alpha- and beta-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry revealed that hemoglobin protein was specifically localized in type II cells of a lung cell mixture and rat lung tissue. The endogenous synthesis of hemoglobin in alveolar epithelial cells suggests that hemoglobin may have unidentified functions other than oxygen transport in the lung.
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Project description:We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present.
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Project description:Inhaled nanoparticles have a high deposition rate in the alveolar units of the deep lung. The alveolar epithelium is composed of type-I and type-II epithelial cells (ATI and ATII respectively) and is bathed in pulmonary surfactant. The effect of native human ATII cell secretions on nanoparticle toxicity is not known. We investigated the cellular uptake and toxicity of silver nanowires (AgNWs; 70 nm diameter, 1.5 μm length) with human ATI-like cells (TT1), in the absence or presence of Curosurf® (a natural porcine pulmonary surfactant with a low amount of protein) or harvested primary human ATII cell secretions (HAS; containing both the complete lipid as well as the full protein complement of human pulmonary surfactant i.e. SP-A, SP-B, SP-C and SP-D). We hypothesised that Curosurf® or HAS would confer improved protection for TT1 cells, limiting the toxicity of AgNWs. In agreement with our hypothesis, HAS reduced the inflammatory and reactive oxygen species (ROS)-generating potential of AgNWs with exposed TT1 cells. For example, IL-8 release and ROS generation was reduced by 38% and 29%, respectively, resulting in similar levels to that of the non-treated controls. However in contrast to our hypothesis, Curosurf® had no effect. We found a significant reduction in AgNW uptake by TT1 cells in the presence of HAS but not Curosurf. Furthermore, we show that the SP-A and SP-D are likely to be involved in this process as they were found to be specifically bound to the AgNWs. While ATI cells appear to be protected by HAS, evidence suggested that ATII cells, despite no uptake, were vulnerable to AgNW exposure (indicated by increased IL-8 release and ROS generation and decreased intracellular SP-A levels one day post-exposure). This study provides unique findings that may be important for the study of lung epithelial-endothelial translocation of nanoparticles in general and associated toxicity within the alveolar unit.
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Project description:Alveolar type II (ATII) cells are essential for the maintenance of the alveolar homeostasis. However, knowledge of the expression of the miRNAs and miRNA-regulated networks which control homeostasis and coordinate diverse functions of murine ATII cells is limited. Therefore, we asked how miRNAs expressed in ATII cells might contribute to the regulation of signaling pathways. We purified "untouched by antibodies" ATII cells using a flow cytometric sorting method with a highly autofluorescent population of lung cells. TaqMan® miRNA low-density arrays were performed on sorted cells and intersected with miRNA profiles of ATII cells isolated according to a previously published protocol. Of 293 miRNAs expressed in both ATII preparations, 111 showed equal abundances. The target mRNAs of bona fide ATII miRNAs were used for pathway enrichment analysis. This analysis identified nine signaling pathways with known functions in fibrosis and/or epithelial-to-mesenchymal transition (EMT). In particular, a subset of 19 miRNAs was found to target 21 components of the TGF-β signaling pathway. Three of these miRNAs (miR-16-5p, -17-5p and -30c-5p) were down-modulated by TGF-β1 stimulation in human A549 cells, and concomitant up-regulation of associated mRNA targets (BMPR2, JUN, RUNX2) was observed. These results suggest an important role for miRNAs in maintaining the homeostasis of the TGF-β signaling pathway in ATII cells under physiological conditions.
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Project description:Annexin A2, a calcium-dependent phospholipid-binding protein, is abundantly expressed in alveolar type II cells where it plays a role in lung surfactant secretion. Nevertheless, little is known about the details of its cellular pathways. To identify annexin A2-regulated or associated proteins, we silenced endogenous annexin A2 expression in rat alveolar type II cells by RNA interference and assessed the change of the cellular transcriptome by DNA microarray analysis. The loss of annexin A2 resulted in the change of 61 genes. Thirteen of the selected genes (11 down-regulated and 2 up-regulated genes) were validated by real time quantitative PCR. When the loss of rat annexin A2 was rescued by overexpressing EGFP-tagged human annexin A2, six of seven selected targets returned to their normal expression level, indicating that these genes are indeed annexin A2-associated targets. One of the targets, Rab14, co-immunoprecipitated with annexin A2. Rab14 also co-localized in part with annexin A2 and lamellar bodies in alveolar type II cells. The silencing of Rab14 resulted in a decrease in surfactant secretion, suggesting that Rab14 may play a role in surfactant secretion.
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Project description:BackgroundLysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.MethodsMouse lung cells were sorted on the basis of CD45(neg)CD31(neg)EpCAM(pos)LysoTracker(pos) expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.ResultsThe purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker(pos) AT2 cells generated SP-C(pos) alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.ConclusionsThis study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.
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Project description:Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5 - /KRT17 + aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.
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Project description:Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells.
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Project description:Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome-and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra-alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia-inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti-inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL-10, while decreasing IL-1 and IL-6 expression. Mice with ATII-specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co-cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti-inflammatory phenotype and dampens excessive inflammation in ALI.
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