RESUMEN
Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.
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Células Epiteliales Alveolares , Mecanotransducción Celular , Células Epiteliales Alveolares/metabolismo , Células Cultivadas , Pulmón , Diferenciación Celular/fisiología , RespiraciónRESUMEN
The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.
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COVID-19/metabolismo , Coronavirus Humano OC43/metabolismo , Proteínas de la Membrana/metabolismo , SARS-CoV-2/metabolismo , Transducción de Señal , Células A549 , Animales , COVID-19/genética , Chlorocebus aethiops , Coronavirus Humano OC43/genética , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , SARS-CoV-2/genética , Células VeroRESUMEN
The mechanistic target of rapamycin (mTOR) and wingless-related integration site (Wnt) signal transduction networks are evolutionarily conserved mammalian growth and cellular development networks. Most cells express many of the proteins in both pathways, and this review will briefly describe only the key proteins and their intra- and extracellular crosstalk. These complex interactions will be discussed in relation to cancer development, drug resistance, and stem cell exhaustion. This review will also highlight the tumor-suppressive tuberous sclerosis complex (TSC) mutated, mTOR-hyperactive lung disease of women, lymphangioleiomyomatosis (LAM). We will summarize recent advances in the targeting of these pathways by monotherapy or combination therapy, as well as future potential treatments.
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Linfangioleiomiomatosis/fisiopatología , Terapia Molecular Dirigida , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Wnt/metabolismo , Animales , Humanos , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/metabolismoRESUMEN
OBJECTIVE: The aim of this study was to identify the factors associated with delays in treatment of sickle pain crisis in the pediatric emergency department with the goal of discerning whether earlier pain management is correlated with better clinical outcome. METHODS: This retrospective study examined data collected from clinical records of patients, aged 21 years or younger, who was treated for sickle cell pain crisis between January and June 2012. Demographic and clinical characteristics were extracted from electronic records, as well as time of registration, triage, initial pain assessment, analgesic administration, and pain reassessment. RESULT: A total of 160 sickle cell pain crises visits by 67 unique patients were identified. Opiates were the most common initial pain medication prescribed and administered. The mean time to initial analgesic administration and pain reassessment was 89 and 60 minutes, respectively. Patients with orders for imaging studies experienced significant delays in time to initial analgesic medication and pain reassessment. In addition, higher triage pain score correlated with shorter time to first dose of pain medication. However, age, sex, and final disposition did not affect time to administration of analgesic medications.Earlier pain management resulted in shorter ED length of stay for all patients regardless of disposition. However, earlier pain management did not affect the total length of hospitalization for patients admitted to the inpatient services. CONCLUSIONS: Pediatric patients with sickle cell pain crises experienced significant delays to initial analgesic medication. A standardized approach to pain management may improve ED management of SCD crises.
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Dolor Agudo/tratamiento farmacológico , Anemia de Células Falciformes/terapia , Mejoramiento de la Calidad , Adolescente , Analgésicos/uso terapéutico , Analgésicos Opioides/uso terapéutico , Niño , Preescolar , Servicio de Urgencia en Hospital , Femenino , Humanos , Lactante , Masculino , Manejo del Dolor/métodos , Estudios Retrospectivos , Adulto JovenRESUMEN
Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.
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Recurrent infections are a hallmark of STAT3 dominant-negative hyper-IgE syndrome (STAT3 HIES), a rare immunodeficiency syndrome previously known as Jobs syndrome, along with elevated IgE levels and impaired neutrophil function. We have been developing nanoparticles with neutrophil trophism that home to the sites of infection via these first-responder leukocytes, named neutrophil-avid nanocarriers (NANs). Here, we demonstrate that human neutrophils can phagocytose nanogels (NGs), a type of NAN, with enhanced uptake after particle serum opsonization, comparing neutrophils from healthy individuals to those with STAT3 HIES, where both groups exhibit NG uptake; however, the patient group showed reduced phagocytosis efficiency with serum-opsonized NANs. Proteomic analysis of NG protein corona revealed complement components, particularly C3, as predominant in both groups. Difference between groups includes STAT3 HIES samples with higher neutrophil protein and lower acute-phase protein expression. The study suggests that despite neutrophil dysfunction in STAT3 HIES, NANs have potential for directed delivery of cargo therapeutics to improve neutrophil infection clearance.
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Síndrome de Job , Nanopartículas , Neutrófilos , Fagocitosis , Factor de Transcripción STAT3 , Humanos , Neutrófilos/metabolismo , Neutrófilos/inmunología , Síndrome de Job/metabolismo , Factor de Transcripción STAT3/metabolismo , Femenino , Masculino , Adulto , Proteómica/métodos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Complemento C3/metabolismoRESUMEN
Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-ß signaling through TGF-ßR2 (transforming growth factor-ß receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelial Tgfbr2 exhibited prolonged injury and diminished vascular repair. Loss of endothelial Tgfbr2 prevented autocrine Vegfa (vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-ßR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-ß signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-ßR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-ßR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection.
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Gripe Humana , Humanos , Ratones , Animales , Receptor Tipo II de Factor de Crecimiento Transformador beta , Factor A de Crecimiento Endotelial Vascular , Pulmón/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , ARN MensajeroRESUMEN
Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.
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Linfangioleiomiomatosis , Proteína 2 del Complejo de la Esclerosis Tuberosa , Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Animales , Humanos , Lactante , Ratones , Células Endoteliales/metabolismo , Pulmón/metabolismo , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mesodermo/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Remodelación Vascular/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Técnicas In VitroRESUMEN
Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.
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Perfilación de la Expresión Génica , Difusión de la Información , Animales , Ratones , Humanos , Análisis de la Célula Individual , TranscriptomaRESUMEN
Plant tolerance to herbivory may depend on local environmental conditions. Models predict both increased and decreased tolerance with increasing resources. Transgenerational effects of herbivory may result in cross-generation tolerance. We evaluated within- and potential between-generation consequences of deer browsing in light-gap and understory habitats in the forest-edge herb, Campanulastrum americanum. Plants were assigned to deer-browsed, simulated-herbivory, and control (undamaged) treatments in the two light environments. In light gaps, plants were eaten earlier, more frequently, and had less vegetative recovery relative to uneaten plants than in the understory. As a result, browsed light-gap plants had a greater reduction in flowers and fruit than understory plants. This reduced tolerance was in part because deer browsing damaged plants in light gaps more than those in the understory. However, in the simulated herbivory treatment, where damage levels were similar between light habitats, plants growing in high-resource light gaps also had reduced tolerance of herbivory relative to those in the forest understory. C. americanum's reproductive phenology was delayed by reduced light and the loss of the apical meristem. As a result, deer-browsed plants in the light gap flowered slightly later than uneaten plants in the understory. C. americanum has a polymorphic life history and maternal flowering time influences the frequency of annual and biennial offspring. The later flowering of deer-browsed plants in light gaps will likely result in a reduced frequency of high-fitness annual offspring and an increase in lower fitness biennial offspring. Therefore, additional between-generation costs of herbivory are expected relative to those predicted by fruit number alone.
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Campanulaceae/fisiología , Ecosistema , Luz Solar , Animales , Ciervos , Conducta Alimentaria , Factores de Tiempo , VirginiaRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. Here, we profile a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing (scRNA-seq) analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of rapamycin-sensitive expression of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM-like phenotype. The alterations in gene expression are driven by distinctive crosstalk between mesenchymal and epithelial subsets of cells observed in mesenchymal Tsc2-deficient lungs. This study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype.
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Pulmón/metabolismo , Linfangioleiomiomatosis/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mesodermo/metabolismo , Factores de Edad , Anciano , Animales , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/fisiopatología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mesodermo/efectos de los fármacos , Ratones , Factores Sexuales , Sirolimus/administración & dosificación , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Vía de Señalización WntRESUMEN
OBJECTIVE: To assess the sensitivity and negative predictive value (NPV) of screening renal and bladder ultrasound (RBUS) after initial febrile urinary tract infection (UTI) among patients with clinically significant vesicoureteral reflux (VUR). METHODS: A retrospective review was performed of all children <2 years of age who presented with a febrile UTI between 2004 and 2011. The sensitivity and NPV of initial RBUS was calculated among patients who were found to have high-grade (IV-V) VUR. Additionally, initial RBUS among patients with evidence of photopenia on dimercaptosuccinic acid (DMSA) scan or who underwent surgical intervention were reviewed. RESULTS: One hundred forty-four patients with febrile UTI were identified; available RBUS, voiding cystourethrogram (VCUG), and DMSA results for each kidney were reviewed. One hundred fifty-eight kidneys had evidence of VUR on VCUG, and initial RBUS demonstrated abnormality in 25 (sensitivity 0.17). Forty-five kidneys had high-grade VUR and RBUS revealed abnormality in 16 (sensitivity 0.36). One hundred seventy-eight kidneys had no evidence of abnormality on initial RBUS, and 136 (76%) were found to have VUR (NPV 0.24), of which 31 had high-grade VUR (NPV 0.83). Seven kidneys had scarring on DMSA and initial RBUS was normal in 4 (57%). Twelve of 19 patients (63%) who eventually underwent surgical intervention had a normal initial RBUS. CONCLUSION: RBUS has poor sensitivity and NPV for detecting high-grade VUR in patients <2 years who present with a febrile UTI. A significant number of patients who were diagnosed with high-grade VUR, renal scarring, or underwent surgical correction of VUR had a negative screening RBUS.
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Infecciones Urinarias/complicaciones , Reflujo Vesicoureteral/complicaciones , Reflujo Vesicoureteral/diagnóstico por imagen , Femenino , Fiebre/complicaciones , Humanos , Lactante , Masculino , Estudios Retrospectivos , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
Many animals use carotenoid pigments derived from their diet for coloration and immunity. The carotenoid trade-off hypothesis predicts that, under conditions of carotenoid scarcity, individuals may be forced to allocate limited carotenoids to either coloration or immunity. In polychromatic species, the pattern of allocation may differ among individuals. We tested the carotenoid trade-off hypothesis in the Midas cichlid, Amphilophus citrinellus, a species with two ontogenetic color morphs, barred and gold, the latter of which is the result of carotenoid expression. We performed a diet-supplementation experiment in which cichlids of both color morphs were assigned to one of two diet treatments that differed only in carotenoid content (beta-carotene, lutein, and zeaxanthin). We measured integument color using spectrometry, quantified carotenoid concentrations in tissue and plasma, and assessed innate immunity using lysozyme activity and alternative complement pathway assays. In both color morphs, dietary carotenoid supplementation elevated plasma carotenoid circulation but failed to affect skin coloration. Consistent with observable differences in integument coloration, we found that gold fish sequestered more carotenoids in skin tissue than barred fish, but barred fish had higher concentrations of carotenoids in plasma than gold fish. Neither measure of innate immunity differed between gold and barred fish, or as a function of dietary carotenoid supplementation. Lysozyme activity, but not complement activity, was strongly affected by body condition. Our data show that a diet low in carotenoids is sufficient to maintain both coloration and innate immunity in Midas cichlids. Our data also suggest that the developmental transition from the barred to gold morph is not accompanied by a decrease in innate immunity in this species.