RESUMEN
Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFß negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.
Asunto(s)
Lesión Pulmonar Aguda , Linaje de la Célula , Fibroblastos , Neumonía , Alveolos Pulmonares , Fibrosis Pulmonar , Animales , Femenino , Humanos , Masculino , Ratones , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/metabolismo , Diferenciación Celular , Fibroblastos/patología , Fibroblastos/metabolismo , Homeostasis , Neumonía/patología , Neumonía/metabolismo , Alveolos Pulmonares/patología , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Nicho de Células Madre , Células Madre/metabolismo , Células Madre/citología , Células Madre/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.
Asunto(s)
Antineoplásicos Inmunológicos , Carcinoma de Células Escamosas , Animales , Anticuerpos Monoclonales , Células CHO , Cricetinae , Cricetulus , Ratones , Mucina-1/química , Mucinas/química , Polisacáridos/químicaRESUMEN
Pulmonary fibrosis is characterized by progressive and irreversible scarring of alveoli, which causes reduction of surface epithelial area and eventually respiratory failure. The precise mechanism of alveolar scarring is poorly understood. In this study, we explored transcriptional signatures of activated fibroblasts in alveolar airspaces by using intratracheal transfer in bleomycin-induced lung fibrosis. Lung fibroblasts transferred into injured alveoli upregulated genes related to translation and metabolism in the first two days, and upregulated genes related to extracellular matrix (ECM) production between day 2 and 7. Upstream analysis of these upregulated genes suggested possible contribution of hypoxia-inducible factors 1a (Hif1a) to fibroblast activation in the first two days, and possible contribution of kruppel-like factor 4 (Klf4) and glioma-associated oncogene (Gli) transcription factors to fibroblast activation in the following profibrotic phase. Fibroblasts purified based on high Acta2 expression after intratracheal transfer were also characterized by ECM production and upstream regulation by Klf4 and Gli proteins. Pharmacological inhibition of Gli proteins by GANT61 in bleomycin-induced lung fibrosis altered the pattern of scarring characterized by dilated airspaces and smaller fibroblast clusters. Activated fibroblasts isolated from GANT61-treated mice showed decreased migration capacity, suggesting that Gli signaling inhibition attenuated fibroblast activation. In conclusion, we revealed transcriptional signatures and possible upstream regulators of activated fibroblasts in injured alveolar airspaces. The altered scar formation by Gli signaling inhibition supports that activated fibroblasts in alveolar airspaces may play a critical role in scar formation.
Asunto(s)
Cicatriz/metabolismo , Cicatriz/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Transducción de Señal , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Cicatriz/genética , Fibroblastos/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones Endogámicos C57BL , Fibrosis Pulmonar/genética , Piridinas/farmacología , Pirimidinas/farmacología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Mice that globally overexpress the transcription factor Fos-related antigen-2 (Fra-2) develop extensive pulmonary fibrosis and pulmonary vascular remodeling. To determine if these phenotypes are a consequence of ectopic Fra-2 expression in vascular smooth muscle cells and myofibroblasts, we generated mice that overexpress Fra-2 specifically in these cell types (α-SMA-rtTA;tetO-Fra-2). Surprisingly, these mice did not develop vascular remodeling or pulmonary fibrosis but did develop a spontaneous emphysema-like phenotype characterized by alveolar enlargement. Secondary septa formation is an important step in the normal development of lung alveoli, and α-smooth muscle actin (SMA)-expressing fibroblasts (myofibroblasts) play a crucial role in this process. The mutant mice showed reduced numbers of secondary septa at postnatal day 7 and enlarged alveolae starting at postnatal day 12, suggesting impairment of secondary septa formation. Lineage tracing using α-SMA-rtTA mice crossed to a floxed TdTomato reporter revealed that embryonic expression of α-SMA Cre marked a population of cells that gave rise to nearly all alveolar myofibroblasts. Comprehensive transcriptome analyses (RNA sequencing) demonstrated that the overwhelming majority of genes whose expression was significantly altered by overexpression of Fra-2 in myofibroblasts encoded secreted proteins, components of the extracellular matrix (ECM), and cell adhesion-associated genes, including coordinate upregulation of pairs of integrins and their principal ECM ligands. In addition, primary myofibroblasts isolated from the mutant mice showed reduced migration capacity. These findings suggest that Fra-2 overexpression might impair myofibroblast functions crucial for secondary septation, such as myofibroblast migration across alveoli, by perturbing interactions between integrins and locally produced components of the ECM.
Asunto(s)
Antígeno 2 Relacionado con Fos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Alveolos Pulmonares/metabolismo , Actinas/metabolismo , Animales , Animales Recién Nacidos , Fibroblastos/metabolismo , Pulmón/metabolismo , Ratones , Fibrosis Pulmonar/metabolismoRESUMEN
Myeloid cells such as monocytes and monocyte-derived macrophages promote tumor progression. Recent reports suggest that extramedullary hematopoiesis sustains a sizable reservoir of tumor-infiltrating monocytes in the spleen. However, the influence of the spleen on tumor development and the extent to which spleen monocytes populate the tumor relative to bone marrow (BM) monocytes remain controversial. Here, we used mice expressing the photoconvertible protein Kikume Green-Red to track the redistribution of monocytes from the BM and spleen, and mice expressing fluorescent ubiquitination-based cell-cycle indicator proteins to monitor active hematopoiesis in these tissues. In mice bearing late-stage tumors, the BM, besides being the major site of monocyte production, supplied the expansion of the spleen reservoir, replacing 9% of spleen monocytes every hour. Deployment of monocytes was equally rapid from the BM and the spleen. However, BM monocytes were younger than those in the spleen and were 2.7 times more likely to migrate into the tumor from the circulation. Partly as a result of this intrinsic difference in migration potential, spleen monocytes made only a minor contribution to the tumor-infiltrating monocyte population. At least 27% of tumor monocytes had traveled from the BM in the last 24 h, compared with only 2% from the spleen. These observations highlight the importance of the BM as the primary hematopoietic tissue and monocyte reservoir in tumor-bearing mice, despite the changes that occur in the spleen monocyte reservoir during tumor development.
Asunto(s)
Células de la Médula Ósea/inmunología , Carcinogénesis/inmunología , Movimiento Celular/inmunología , Hematopoyesis/fisiología , Monocitos/inmunología , Bazo/citología , Análisis de Varianza , Animales , Fluorescencia , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/inmunologíaRESUMEN
Pulmonary fibrosis is a devastating disease for which there are few effective therapies. Activated fibroblasts form subepithelial clusters known as fibroblastic foci, which are characterized by excessive collagen deposition. The origin of activated fibroblasts is controversial and needs to be clarified to understand their pathogenicity. Here, using an intratracheal adoptive cell transfer method, we show that resident fibroblasts in alveolar walls have the highest profibrotic potential. By using collagen I(α)2-green fluorescent protein and neural/glial antigen 2-DsRed fluorescent reporter mice, we identified resident fibroblasts and pericytes in the alveolar walls based on surface marker expression and ultrastructural characteristics. In the early phase of bleomycin-induced pulmonary fibrosis, activated fibroblasts migrated into epithelium-denuded alveolar airspaces. Purified resident fibroblasts delivered into injured alveoli by an intratracheal route showed similar activated signatures as activated fibroblasts and formed fibroblastic foci. Neither pericytes nor epithelial cells had the same profibrotic potential. Transferred resident fibroblasts highly up-regulated profibrotic genes including α-smooth muscle actin and were a significant source of collagen deposition. These data provide insights into the cellular mechanisms of fibrogenesis and show intratracheal cell transfer to be a useful tool for exploring novel therapeutic targets against pulmonary fibrosis.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Fibrosis Pulmonar/patología , Animales , Bleomicina/efectos adversos , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio/metabolismo , Epitelio/patología , Femenino , Fibroblastos/metabolismo , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/metabolismo , Regulación hacia ArribaRESUMEN
Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses in vivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuse lesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, and Fgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.
Asunto(s)
Macrófagos Alveolares/patología , Fibrosis Pulmonar/patología , Receptores CCR2/metabolismo , Animales , Bleomicina/efectos adversos , Modelos Animales de Enfermedad , Células Epiteliales/patología , Perfilación de la Expresión Génica , Humanos , Hidroxiprolina/análisis , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Monocitos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fibrosis Pulmonar/inducido químicamente , Receptores CCR2/genética , Dióxido de Silicio/efectos adversosRESUMEN
Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.
Asunto(s)
Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Animales , Apoptosis , Biomarcadores/metabolismo , Bleomicina , Células de la Médula Ósea/patología , Proliferación Celular , Colágeno Tipo I/metabolismo , Perfilación de la Expresión Génica , Ontología de Genes , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Osteopontina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.
Asunto(s)
Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Fibroblastos , Fibrosis Pulmonar Idiopática , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Ratones , Humanos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/genética , Senoterapéuticos/farmacología , Masculino , Pulmón/patología , Pulmón/metabolismo , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genéticaRESUMEN
Monocyte-derived macrophages recruited to injured tissues induce a maladaptive fibrotic response characterized by excessive production of collagen by local fibroblasts. Macrophages initiate this programming via paracrine factors, but it is unknown whether reciprocal responses from fibroblasts enhance profibrotic polarization of macrophages. We identify macrophage-fibroblast crosstalk necessary for injury-associated fibrosis, in which macrophages induced interleukin 6 ( IL-6 ) expression in fibroblasts via purinergic receptor P2rx4 signaling, and IL-6, in turn, induced arginase 1 ( Arg1 ) expression in macrophages. Arg1 contributed to fibrotic responses by metabolizing arginine to ornithine, which fibroblasts used as a substrate to synthesize proline, a uniquely abundant constituent of collagen. Imaging of idiopathic pulmonary fibrosis (IPF) lung samples confirmed expression of ARG1 in myeloid cells, and arginase inhibition suppressed collagen expression in cultured precision-cut IPF lung slices. Taken together, we define a circuit between macrophages and fibroblasts that facilitates cross-feeding metabolism necessary for injury-associated fibrosis.
RESUMEN
Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFß) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFß signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFß signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.
RESUMEN
Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.
Asunto(s)
Anticuerpos , Fibrosis Pulmonar , Humanos , Fibroblastos , Hepatocitos , CinéticaRESUMEN
Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.
Asunto(s)
Anticuerpos Monoclonales , Carcinoma de Células Escamosas/diagnóstico , Epítopos/análisis , Neoplasias Esofágicas/diagnóstico , Glicoproteínas de Membrana/química , Mucinas/química , Acetilgalactosamina/química , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Células CHO , Secuencia de Carbohidratos , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Cricetinae , Células Epiteliales/química , Células Epiteliales/citología , Epítopos/inmunología , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Esófago/química , Esófago/citología , Citometría de Flujo , Expresión Génica , Glicosilación , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Mucinas/genética , Mucinas/inmunología , TransfecciónRESUMEN
A little-appreciated feature of early pregnancy is that embryo implantation and placental outgrowth do not evoke wound-healing responses in the decidua, the specialized endometrial tissue that surrounds the conceptus. Here, we provide evidence that this phenomenon is partly due to an active program of gene silencing mediated by EZH2, a histone methyltransferase that generates repressive histone 3 lysine 27 trimethyl (H3K27me3) histone marks. We find that pregnancies in mice with EZH2-deficient decidual stromal cells frequently fail by mid-gestation, with the decidua showing ectopic myofibroblast formation, peri-embryonic collagen deposition, and gene expression profiles associated with transforming growth factor ß (TGF-ß)-driven fibroblast activation and fibrogenic extracellular matrix (ECM) remodeling. Analogous responses are observed when the mutant decidua is surgically wounded, while blockade of TGF-ß receptor signaling inhibits the defects and improves reproductive outcomes. Together, these results highlight a critical feature of reproductive success and have implications for the context-specific control of TGF-ß-mediated wound-healing responses elsewhere in the body.
Asunto(s)
Implantación del Embrión/fisiología , Silenciador del Gen/fisiología , Placenta/metabolismo , Factor de Crecimiento Transformador beta/genética , Cicatrización de Heridas/fisiología , Animales , Decidua/metabolismo , Embrión de Mamíferos/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Expresión Génica/fisiología , Histonas/metabolismo , Humanos , Ratones Endogámicos C57BL , Embarazo , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.
RESUMEN
Macrophages are paracrine signalers that regulate tissular responses to injury through interactions with parenchymal cells. Connexin hemichannels have recently been shown to mediate efflux of ATP by macrophages, with resulting cytosolic calcium responses in adjacent cells. Here we report that lung macrophages with deletion of connexin 43 (MacΔCx43) had decreased ATP efflux into the extracellular space and induced a decreased cytosolic calcium response in co-cultured fibroblasts compared to WT macrophages. Furthermore, MacΔCx43 mice had decreased lung fibrosis after bleomycin-induced injury. Interrogating single cell data for human and mouse, we found that P2rx4 was the most highly expressed ATP receptor and calcium channel in lung fibroblasts and that its expression was increased in the setting of fibrosis. Fibroblast-specific deletion of P2rx4 in mice decreased lung fibrosis and collagen expression in lung fibroblasts in the bleomycin model. Taken together, these studies reveal a Cx43-dependent profibrotic effect of lung macrophages and support development of fibroblast P2rx4 as a therapeutic target for lung fibrosis.
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Conexina 43 , Fibrosis Pulmonar Idiopática , Adenosina Trifosfato/metabolismo , Animales , Bleomicina/farmacología , Calcio/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Macrófagos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.
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Inmunidad , Inmunoterapia , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Antineoplásicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Granzimas/metabolismo , Interferón gamma/metabolismo , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Transducción de Señal , Proteína smad3/metabolismo , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Aberrant epithelial reprogramming can induce metaplastic differentiation at sites of tissue injury that culminates in transformed barriers composed of scar and metaplastic epithelium. While the plasticity of epithelial stem cells is well characterized, the identity and role of the niche has not been delineated in metaplasia. Here, we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts during scarring, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation signalling to upregulate BMP antagonism in the progenitor niche that promotes metaplasia. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation into SFTPC+ epithelium. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signalling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.
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Células Epiteliales Alveolares/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fibrosis Pulmonar/metabolismo , Nicho de Células Madre , Proteína con Dedos de Zinc GLI1/metabolismo , Células Epiteliales Alveolares/patología , Animales , Bleomicina , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Proteínas Hedgehog/metabolismo , Queratina-5/metabolismo , Pulmón/patología , Células Madre Mesenquimatosas/patología , Metaplasia , Ratones Endogámicos C57BL , Ratones Noqueados , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Transducción de Señal , Receptor Smoothened/metabolismo , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Espacio Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Drosophila/metabolismo , Estrés del Retículo Endoplásmico , Evolución Molecular , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/química , Filogenia , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Interferencia de ARN , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1-expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1-expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis.