RESUMEN

BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

Asunto(s)

Diferenciación Celular , Pulmón , Organoides , Humanos , Organoides/citología , Organoides/metabolismo , Pulmón/citología , Pulmón/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Técnicas de Cocultivo/métodos , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo

RESUMEN

While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq, bulk ATAC-seq, and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.

Asunto(s)

Células Epiteliales Alveolares , Diferenciación Celular , Cromatina , Proteínas de Unión al ADN , Factor Nuclear Tiroideo 1 , Factores de Transcripción , Animales , Factor Nuclear Tiroideo 1/metabolismo , Factor Nuclear Tiroideo 1/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cromatina/metabolismo , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Ratones Noqueados , Pulmón/citología , Pulmón/metabolismo , Linaje de la Célula , Femenino

RESUMEN

Major histocompatibility complex class I (MHC I) molecules present peptides to CD8+ T-cells for immunosurveillance of infection and cancer. Recent studies indicate lineage-specific heterogeneity in MHC I expression. While respiratory diseases rank among the leading causes of mortality, studies in mice have shown that lung epithelial cells (LECs) express the lowest levels of MHC I in the lung. This study aims to answer three questions: (i) Do human LECs express low levels of MHC I? (ii) Is LEC MHC I expression modulated in chronic respiratory diseases? (iii) Which factors regulate MHC I levels in human LECs? We analyzed human LECs from parenchymal explants using single-cell RNA sequencing and immunostaining. We confirmed low constitutive MHC I expression in human LECs, with significant upregulation in chronic respiratory diseases. We observed a sexual dimorphism, with males having higher MHC I levels under steady-state conditions, likely due to differential redox balance. Our study unveils the complex interplay between MHC I expression, sex, and respiratory disease. Since MHC I upregulation contributes to the development of immunopathologies in other models, we propose that it may have a similar impact on chronic lung disease.

Asunto(s)

Células Epiteliales , Antígenos de Histocompatibilidad Clase I , Pulmón , Humanos , Femenino , Masculino , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Pulmón/metabolismo , Pulmón/citología , Pulmón/inmunología , Células Epiteliales/metabolismo , Caracteres Sexuales , Enfermedades Pulmonares/metabolismo

RESUMEN

BACKGROUND: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. METHODS: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-ß phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. RESULTS: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFß signalling inhibition and BMP pathway activation. CONCLUSIONS: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation.

Asunto(s)

Diferenciación Celular , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Pulmón , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Pulmón/metabolismo , Pulmón/embriología , Pulmón/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología

RESUMEN

Short-lived repair-promoting macrophages are recruited to foci of lung damage during influenza infection-and they are Ly6G positive (see related Research Article by Ruscitti et al.).

Asunto(s)

Antígenos Ly , Macrófagos Alveolares , Animales , Antígenos Ly/metabolismo , Antígenos Ly/inmunología , Ratones , Macrófagos Alveolares/inmunología , Pulmón/inmunología , Pulmón/citología , Biomarcadores , Macrófagos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Humanos

RESUMEN

BACKGROUND: Lung fibroblasts play a central role in maintaining lung homeostasis and facilitating repair through the synthesis and organization of the extracellular matrix (ECM). This study investigated the cross-talk between interleukin-1 alpha (IL-1α) and transforming growth factor-ß (TGF-ß) signaling, two key regulators in tissue repair and fibrosis, in the context of lung fibroblast repair in the healthy lung. RESULTS: Stimulation of lung fibroblasts with TGF-ß1 and TGF-ß2 induced collagen-I and fibronectin protein expression (p < 0.05), a response inhibited with co-treatment with IL-1α (p < 0.05). Additionally, TGF-ß1 and TGF-ß2 induced myofibroblast differentiation, and collagen-I gel contraction, which were both suppressed by IL-1α (p < 0.05). In contrast, interleukin (IL)-6, IL-8 and thymic stromal lymphopoietin induced by IL-1α, were unaffected by TGF-ß1 or TGF-ß2. Mechanistically, IL-1α administration led to the suppression of TGF-ß1 and TGF-ß2 signaling, through downregulation of mRNA and protein for TGF-ß receptor II and the downstream adaptor protein TRAF6, but not through miR-146a that is known to be induced by IL-1α. DISCUSSION: IL-1α acts as a master regulator, modulating TGF-ß1 and TGF-ß2-induced ECM production, remodeling, and myofibroblast differentiation in human lung fibroblasts, playing a vital role in balancing tissue repair versus fibrosis. Further research is required to understand the dysregulated cross-talk between IL-1α and TGF-ß signaling in chronic lung diseases and the exploration of therapeutic opportunities. METHODS: Primary human lung fibroblasts (PHLF) were treated with media control, or 1 ng/ml IL-1α with or without 50 ng/ml TGF-ß1 or TGF-ß2 for 1, 6 and 72 h. Cell lysates were assessed for the expression of ECM proteins and signaling molecules by western blot, miRNA by qPCR, mRNA by RNA sequencing and cell supernatants for cytokine production by ELISA. PHLFs were also seeded in non-tethered collagen-I gels to measure contraction, and myofibroblast differentiation using confocal microscopy.

Asunto(s)

Matriz Extracelular , Fibroblastos , Interleucina-1alfa , Pulmón , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Humanos , Interleucina-1alfa/metabolismo , Interleucina-1alfa/genética , Matriz Extracelular/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Pulmón/metabolismo , Pulmón/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/citología , Diferenciación Celular , Miofibroblastos/metabolismo , Miofibroblastos/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , MicroARNs/genética , MicroARNs/metabolismo , Fibronectinas/metabolismo , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Transformador beta2

RESUMEN

In this work, a benzofuranone-derived fluorescent probe BFSF was developed for imaging the sulphite level in living hypoxia pulmonary cells. Under the excitation of 510 nm, BFSF showed a strong fluorescence response at 570 nm when reacted with sulphite. In the solution system, the constructed hypercapnia and serious hypercapnia conditions did not affect the fluorescence response. In comparison with the recently reported probes, BFSF suggested the advantages including rapid response, steady signal reporting, high specificity and low cytotoxicity upon living lung cells. Under a normal incubation atmosphere, BFSF realized the imaging of both exogenous and endogenous sulphite in living pulmonary cells. In particular, BFSF achieved imaging the decrease of the sulphite level under severe hypoxia as well as the recovery of the sulphite level with urgent oxygen supplement. With the imaging capability for the sulphite level in living pulmonary cells under hypoxia conditions, BFSF together with the information herein was meaningful for investigating the anaesthesia-related biological indexes.

Asunto(s)

Benzofuranos , Colorantes Fluorescentes , Pulmón , Sulfitos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Benzofuranos/química , Benzofuranos/síntesis química , Sulfitos/análisis , Sulfitos/química , Pulmón/diagnóstico por imagen , Pulmón/citología , Humanos , Hipoxia de la Célula , Imagen Óptica , Estructura Molecular

RESUMEN

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection. As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Asunto(s)

Pulmón , Animales , Ratones , Pulmón/inmunología , Pulmón/citología , Péptidos/inmunología , Péptidos/química , Linfocitos T/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Epítopos de Linfocito T/inmunología

RESUMEN

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

Asunto(s)

Anticuerpos Monoclonales , Cricetulus , Glucosa , Animales , Glucosa/metabolismo , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Cricetinae , Línea Celular , Medio de Cultivo Libre de Suero , Metabolómica/métodos , Pulmón/metabolismo , Pulmón/citología , Metaboloma , Inmunoglobulina G/metabolismo , Células CHO , Técnicas de Cultivo Celular por Lotes/métodos , Glutamina/metabolismo

RESUMEN

Despite significant advancements in cancer treatment in recent decades, the high mortality rate associated with lung cancer remains a significant concern. The development and proper execution of new targeted therapies needs more deep knowledge regarding the lung cancer associated tumour microenvironment. One of the key component of that tumour microenvironment is the lung resident macrophages. Although in normal physiological condition the lung resident macrophages are believed to maintain lung homeostasis, but they may also initiate a vicious inflammatory response in abnormal conditions which is linked to lung cancer development. Depending on the activation pathway, the lung resident macrophages are either of M1 or M2 sub-type. The M1 and M2 sub-types differ significantly in various prospectuses, from phenotypic markers to metabolic pathways. In addition to this generalized classification, the recent advancement of the multiomics technology is able to identify some other sub-types of lung resident macrophages. Researchers have also observed that these different sub-types can manipulate the pathogenesis of lung carcinogenesis in a context dependent manner and can either promote or inhibit the development of lung carcinogenesis upon receiving proper activation. As proper knowledge about the role played by the lung resident macrophages' in shaping the lung carcinogenesis is limited, so the main purpose of this review is to bring all the available information under the same roof. We also elaborated the different mechanisms involved in maintenance of the plasticity of M1/M2 sub-type, as this plasticity can be a good target for lung cancer treatment.

Asunto(s)

Carcinogénesis , Neoplasias Pulmonares , Macrófagos , Humanos , Animales , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Macrófagos/metabolismo , Pulmón/citología , Pulmón/fisiología , Macrófagos Alveolares/metabolismo , Progresión de la Enfermedad

RESUMEN

3D printing can revolutionize personalized medicine by allowing cost-effective, customized tissue-engineering constructs. However, the limited availability and diversity of biopolymeric hydrogels restrict the variety and applications of bioinks. In this study, we introduce a composite bioink for 3D bioprinting, combining a photo-cross-linkable derivative of Mucin (Mu) called Methacrylated Mucin (MuMA) and Hyaluronic acid (HA). The less explored Mucin is responsible for the hydrogel nature of mucus and holds the potential to be used as a bioink material because of its plethora of features. HA, a crucial extracellular matrix component, is mucoadhesive and enhances ink viscosity and printability. Photo-cross-linking with 405 nm light stabilizes the printed scaffolds without damaging cells. Rheological tests reveal shear-thinning behavior, aiding cell protection during printing and improved MuMA bioink viscosity by adding HA. The printed structures exhibited porous behavior conducive to nutrient transport and cell migration. After 4 weeks in phosphate-buffered saline, the scaffolds retain 70% of their mass, highlighting stability. Biocompatibility tests with lung epithelial cells (L-132) confirm cell attachment and growth, suggesting suitability for lung tissue engineering. It is envisioned that the versatility of bioink could lead to significant advancements in lung tissue engineering and various other biomedical applications.

Asunto(s)

Materiales Biocompatibles , Bioimpresión , Ácido Hialurónico , Ensayo de Materiales , Mucinas , Impresión Tridimensional , Ingeniería de Tejidos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Humanos , Mucinas/química , Mucinas/metabolismo , Tinta , Luz , Pulmón/citología , Tamaño de la Partícula , Andamios del Tejido/química , Hidrogeles/química , Hidrogeles/farmacología

RESUMEN

Mesenchymal stem cells (MCSs) secretome provide MSC-like therapeutic effects in preclinical models of lung injury, circumventing safety concerns with the use of live cells. Secretome consists of Extracellular Vesicles (EVs), including populations of nano- to micro-sized particles (exosomes and microvesicles) delimited by a phospholipidic bilayer. However, its poor stability and bioavailability severely limit its application. The role of Hyaluronic acid (HA) as potential carrier in biomedical applications has been widely demonstrated. Here, we investigated the interplay between HA and MSCs- secretome blends and their ability to exert a bioactive effect on pulmonary differentiation in a 3D microenvironment mimicking lung niche. To this aim, the physical-chemical properties of HA/Secre blends have been characterized at low, medium and high HA Molecular Weights (MWs), by means of SEM/TEM, DLS, confocal microscopy and FTIR. Collectively physical-chemical properties highlight the interplay between the HA and the EVs. In 3D matrices, HA/Secre blends showed to promote differentiation in pulmonary lineage, improved as the MW of the HA in the blends decreased. Finally, HA/Secre blends' ability to cross an artificial mucus has been demonstrated. Overall, this work provides new insights for the development of future devices for the therapy of respiratory diseases that are still unmet.

Asunto(s)

Diferenciación Celular , Ácido Hialurónico , Pulmón , Células Madre Mesenquimatosas , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Humanos , Pulmón/metabolismo , Pulmón/citología , Secretoma/metabolismo , Biomimética/métodos , Microambiente Celular/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química

RESUMEN

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-ß1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-ß1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-ß1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-ß1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-ß1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.

Asunto(s)

Diferenciación Celular , Fibroblastos , Fibrosis Pulmonar Idiopática , Miofibroblastos , Factor de Crecimiento Transformador beta1 , Humanos , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Línea Celular , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Pulmón/patología , Pulmón/citología , Transcriptoma , Metformina/farmacología , Plasticidad de la Célula/efectos de los fármacos , Fenotipo

RESUMEN

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Asunto(s)

Enterovirus , Fibroblastos , Aguas Residuales , Humanos , Enterovirus/genética , Enterovirus/aislamiento & purificación , Aguas Residuales/virología , Fibroblastos/virología , Guatemala/epidemiología , Pulmón/virología , Pulmón/citología , Epidemiología Molecular , Línea Celular , Filogenia , Animales , Poliovirus/genética , Poliovirus/aislamiento & purificación , Aguas del Alcantarillado/virología , Ratones , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/epidemiología

RESUMEN

Ex vivo 3D culture of human tissue explants addresses many limitations of traditional monolayer cell culture techniques, namely the lack of cellular heterogeneity and absence of 3D intercellular spatial relationships, but presents challenges with regard to repeatability owing to the difficulty of acquiring multiple tissue samples from the same donor. In this study, we used a cryopreserved bank of human lung microexplants, ∼1 mm3 fragments of peripheral lung from donors undergoing lung resection surgery, and a liquid-like solid 3D culture matrix to describe a method for the analysis of non-small-cell lung cancer adhesion to human lung tissue. H226 (squamous cell carcinoma), H441 (lung adenocarcinoma), and H460 (large cell carcinoma) cell lines were cocultured with lung microexplants. Confocal fluorescence microscopy was used to visualize the adherence of each cell line to lung microexplants. Adherent cancer cells were quantified following filtration of nonadherent cells, digestion of cultured microexplants, and flow cytometry. This method was used to evaluate the role of integrins in cancer cell adherence. A statistically significant decrease in the adherence of H460 cells to lung microexplants was observed when anti-integrins were administered to H460 cells before coculture with lung microexplants.

Asunto(s)

Adhesión Celular , Neoplasias Pulmonares , Pulmón , Humanos , Neoplasias Pulmonares/patología , Línea Celular Tumoral , Pulmón/patología , Pulmón/citología , Técnicas de Cultivo Tridimensional de Células/métodos , Técnicas de Cocultivo/métodos , Carcinoma de Pulmón de Células no Pequeñas/patología , Integrinas/metabolismo

RESUMEN

The lung is a highly mechanical organ as it is exposed to approximately 109 strain cycles, (where strain is the length change of tissue structure per unit initial length), with an approximately 4% amplitude change during quiet tidal breathing or 107 strain cycles at a 25% amplitude during heavy exercises, sighs, and deep inspirations. These mechanical indices have been reported to become aberrant in lung diseases such as acute respiratory distress syndrome (ARDS), pulmonary hypertension, bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), and asthma. Through recent innovations, various in vitro systems/bioreactors used to mimic the lung's mechanical strain have been developed. Among these, the Flexcell tension system which is composed of bioreactors that utilize a variety of programs in vitro to apply static and cyclic strain on different cell-types established as 2D monolayer cultures or cell-embedded 3D hydrogel models, has enabled the assessment of the response of different cells such as fibroblasts to the lung's mechanical strain in health and disease. Fibroblasts are the main effector cells responsible for the production of extracellular matrix (ECM) proteins to repair and maintain tissue homeostasis and are implicated in the excessive deposition of matrix proteins that leads to lung fibrosis. In this review, we summarise, studies that have used the Flexcell tension bioreactor to assess effects of the mechanical lung on the structure, function, and phenotype of lung fibroblasts in homeostatic conditions and abnormal environments associated with lung injury and disease. We show that these studies have revealed that different strain conditions regulate fibroblast proliferation, ECM protein production, and inflammation in normal repair and the diseased lung.

Asunto(s)

Reactores Biológicos , Fibroblastos , Pulmón , Fenotipo , Humanos , Fibroblastos/fisiología , Pulmón/citología , Pulmón/fisiología , Estrés Mecánico , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/patología

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

Asunto(s)

Células Epiteliales Alveolares , Bleomicina , Senescencia Celular , Fibroblastos , Fibrosis Pulmonar Idiopática , Humanos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Bleomicina/toxicidad , Bleomicina/farmacología , Senescencia Celular/efectos de los fármacos , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Medios de Cultivo Condicionados/farmacología , Indoles/farmacología , Fenotipo Secretor Asociado a la Senescencia/efectos de los fármacos , Pulmón/patología , Pulmón/citología , Pulmón/efectos de los fármacos , Sulfonamidas/farmacología , Senoterapéuticos/farmacología , Células Cultivadas , Pirimidinas/farmacología , Pirazoles/farmacología , Nitrilos/farmacología , Compuestos de Anilina

RESUMEN

Fine particulate matters-PM2.5 in the air can have considerable negative effects on human health and the environment. Various human cell-based studies examined the effect of PM2.5 on human health in different cities of the world using various chemical parameters. Unfortunately, limited information is available regarding the relationship between toxicity and chemical characteristics of PM2.5 collected in Istanbul, Türkiye, located in one of the most populated cities in the world. To investigate the chemical characteristics and cytotoxicity of PM2.5 in Istanbul, samples were collected for 12 months, then potentially toxic metals, oxidative potential, and particle indicators (e.g., functional groups and elements) were determined, and the cytotoxicity of PM2.5 on human A549 lung alveolar epithelial cells was examined. The mean PM2.5 mass concentration was 24.0 ± 17.4 µg m-3 and higher in cold months compared to other seasons. Moreover, the results of the metals, elemental, and functional groups indicated that seasonal and monthly characteristics were influenced by the regional anthropogenic sources and photochemistry input. The cytotoxicity results also showed that the viability of A549 cells was reduced with the exposure of PM2.5 (30-53%) and higher cytotoxicity was obtained in summer compared to the other seasons due to the impact of the metals, elements, and oxidative characteristics of PM2.5.

Asunto(s)

Contaminantes Atmosféricos , Supervivencia Celular , Células Epiteliales , Material Particulado , Estaciones del Año , Humanos , Material Particulado/toxicidad , Material Particulado/análisis , Células A549 , Supervivencia Celular/efectos de los fármacos , Contaminantes Atmosféricos/toxicidad , Contaminantes Atmosféricos/análisis , Células Epiteliales/efectos de los fármacos , Turquía , Tamaño de la Partícula , Pulmón/efectos de los fármacos , Pulmón/citología , Monitoreo del Ambiente

RESUMEN

Aging is the main risk factor for chronic lung diseases (CLDs) including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Accordingly, hallmarks of aging like cellular senescence are increased in these patients in different lung cell types including fibroblasts. However, little is known about the different triggers that induce a senescence phenotype in different disease backgrounds and its role in CLD pathogenesis. Therefore, we characterized senescence in primary human lung fibroblasts (phLF) from control, IPF, or COPD patients at baseline and after exposure to disease-relevant insults (H2O2, bleomycin, TGF-ß1) and studied their capacity to support progenitor cell potential in a lung organoid model. Bulk-RNA sequencing revealed that phLF from IPF and COPD activate different transcriptional programs but share a similar senescence phenotype at baseline. Moreover, H2O2 and bleomycin but not TGF-ß1 induced senescence in phLF from different disease origins. Exposure to different triggers resulted in distinct senescence programs in phLF characterized by different SASP profiles. Finally, co-culture with bleomycin- and H2O2-treated phLF reduced the progenitor cell potential of alveolar epithelial progenitor cells. In conclusion, phLF from COPD and IPF share a conserved senescence response that varies depending on the insult and impairs alveolar epithelial progenitor capacity ex vivo.

Asunto(s)

Bleomicina , Senescencia Celular , Fibroblastos , Peróxido de Hidrógeno , Fibrosis Pulmonar Idiopática , Pulmón , Células Madre , Humanos , Senescencia Celular/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/citología , Pulmón/patología , Bleomicina/farmacología , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Células Madre/citología , Peróxido de Hidrógeno/farmacología , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos , Células Cultivadas

RESUMEN

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-ß production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-ß in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-ß production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-ß in fibroblasts, with potential regulation by miRNA-425-5p.

Asunto(s)

Vesículas Extracelulares , Fibroblastos , Pulmón , Macrófagos , MicroARNs , Factor de Crecimiento Transformador beta , MicroARNs/genética , MicroARNs/metabolismo , Fibroblastos/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Factor de Crecimiento Transformador beta/metabolismo , Macrófagos/metabolismo , Pulmón/metabolismo , Pulmón/citología , Humanos , Activación de Macrófagos/genética , Células Cultivadas , Regulación de la Expresión Génica