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There is a growing body of research on the neural control of immunity and inflammation. However, it is not known whether the nervous system can regulate the production of inflammatory myeloid cells from hematopoietic progenitor cells in disease conditions. Myeloid cell numbers in diabetic patients were strongly correlated with plasma concentrations of norepinephrine, suggesting the role of sympathetic neuronal activation in myeloid cell production. The spleens of diabetic patients and mice contained higher numbers of tyrosine hydroxylase (TH)-expressing leukocytes that produced catecholamines. Granulocyte macrophage progenitors (GMPs) expressed the ß2 adrenergic receptor, a target of catecholamines. Ablation of splenic sympathetic neuronal signaling using surgical, chemical, and genetic approaches diminished GMP proliferation and myeloid cell development. Finally, mice lacking TH-producing leukocytes had reduced GMP proliferation, resulting in diminished myelopoiesis. Taken together, our study demonstrates that catecholamines produced by leukocytes and sympathetic nerve termini promote GMP proliferation and myeloid cell development.
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Diabetes Mellitus/fisiopatología , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Mielopoyesis , Neuroinmunomodulación , Sistema Nervioso Simpático/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Leucocitos/enzimología , Leucocitos/metabolismo , Masculino , Ratones , Células Mieloides/citología , Mielopoyesis/efectos de los fármacos , Neuroinmunomodulación/efectos de los fármacos , Norepinefrina/sangre , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/inervación , Bazo/metabolismo , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell-mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-γ) responses along with elevated CD4+NKG7+ and CD4+CD107a+ responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection.
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BACKGROUND: Pulmonary hypertension (PH) is a common complication of systemic sclerosis (SSc) and a leading cause of mortality among patients with this disease. PH can also occur as an idiopathic condition (idiopathic pulmonary arterial hypertension). Investigation of transcriptomic alterations in vascular populations is critical to elucidating cellular mechanisms underlying pathobiology of SSc-associated and idiopathic PH. METHODS: We analyzed single-cell RNA sequencing profiles of endothelial and perivascular mesenchymal populations from explanted lung tissue of patients with SSc-associated PH (n=16), idiopathic pulmonary arterial hypertension (n=3), and healthy controls (n=15). Findings were validated by immunofluorescence staining of explanted human lung tissue. RESULTS: Three disease-associated endothelial populations emerged. Two angiogenic endothelial cell (EC) subtypes markedly expanded in SSc-associated PH lungs: tip ECs expressing canonical tip markers PGF and APLN and phalanx ECs expressing genes associated with vascular development, endothelial barrier integrity, and Notch signaling. Gene regulatory network analysis suggested enrichment of Smad1 (SMAD family member 1) and PPAR-γ (peroxisome proliferator-activated receptor-γ) regulon activities in these 2 populations, respectively. Mapping of potential ligand-receptor interactions highlighted Notch, apelin-APJ (apelin receptor), and angiopoietin-Tie (tyrosine kinase with immunoglobulin-like and EGF-like domains 1) signaling pathways between angiogenic ECs and perivascular cells. Transitional cells, expressing both endothelial and pericyte/smooth muscle cell markers, provided evidence for the presence of endothelial-to-mesenchymal transition. Transcriptional programs associated with arterial endothelial dysfunction implicated VEGF-A (vascular endothelial growth factor-A), TGF-ß1 (transforming growth factor beta-1), angiotensin, and TNFSF12 (tumor necrosis factor ligand superfamily member 12)/TWEAK (TNF-related weak inducer of apoptosis) in the injury/remodeling phenotype of PH arterial ECs. CONCLUSIONS: These data provide high-resolution insights into the complexity and plasticity of the pulmonary endothelium in SSc-associated PH and idiopathic pulmonary arterial hypertension and provide direct molecular insights into soluble mediators and transcription factors driving PH vasculopathy.
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Neovascularización Patológica , Esclerodermia Sistémica , Remodelación Vascular , Humanos , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/complicaciones , Masculino , Femenino , Persona de Mediana Edad , Hipertensión Pulmonar Primaria Familiar/metabolismo , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Hipertensión Pulmonar Primaria Familiar/patología , Estudios de Casos y Controles , Células Endoteliales/metabolismo , Células Endoteliales/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Transcriptoma , Transducción de Señal , Adulto , Análisis de la Célula Individual , Pulmón/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Redes Reguladoras de Genes , AngiogénesisRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a fibrotic age-related chronic lung disease characterized by the accumulation of senescent cells. Whether impaired immune response is responsible for the accumulation of senescent cells in the IPF lung remains unknown. In this study, we characterized the NK phenotype in IPF lungs via flow cytometry using 5-dodecanoylaminofluorescein di-ß-d-galactopyranoside, markers of tissue residence, and chemokine receptors. The effect of the lung microenvironment was evaluated using lung fibroblast (LF) conditioned media (CM), and the bleomycin-induced pulmonary fibrosis mouse model was used to assess the in vivo relationship between NK cells and the accumulation of senescent cells. We found that NK cells from the lower lobe of IPF patients exhibited immune-senescent and impaired CD57-NKG2A+ phenotype. We also observed that culture of NK cells from healthy donors in CM from IPF lower lobe lung fibroblasts induced a senescent-like phenotype and impaired cytotoxic capacity. There is an impaired NK recruitment by LF, and NKs presented decreased migration toward their CM. In addition, NK cell-depleted mice treated with bleomycin showed increased collagen deposition and accumulation of different populations of senescent cells compared with controls. The IPF lung microenvironment induces a dysfunctional NK phenotype limiting the clearance of lung senescent cells and the resolution of lung fibrosis. We propose that impaired NK activity could be one of the mechanisms responsible for perpetuating the accumulation of senescent cells in IPF lungs.
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Antineoplásicos , Fibrosis Pulmonar Idiopática , Ratones , Animales , Pulmón/patología , Fibrosis Pulmonar Idiopática/inducido químicamente , Bleomicina/efectos adversos , Fibrosis , Antineoplásicos/farmacología , FibroblastosRESUMEN
Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.
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RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.
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Fibrosis Pulmonar Idiopática , Animales , Humanos , Ratones , Bleomicina , Quimiocina CXCL6/metabolismo , Quimiocinas/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/genética , Pulmón/patologíaRESUMEN
OBJECTIVE: Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. METHODS: Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. RESULTS: Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. CONCLUSIONS: SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.
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Enfermedades Pulmonares Intersticiales , Esclerodermia Sistémica , Linfocitos T Reguladores , Humanos , Anfirregulina , Linfocitos T CD8-positivos , Células Asesinas Naturales , Pulmón , Enfermedades Pulmonares Intersticiales/inmunología , Células T de Memoria , Esclerodermia Sistémica/inmunologíaRESUMEN
Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c â C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.
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Proteínas F-Box , Animales , Ratones , Abatacept , Aloinjertos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Rechazo de Injerto , Pulmón/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , ARN Mensajero , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a clinical syndrome manifesting with cystic lung disease and pneumothorax. Features of BHD result from the loss-of-function mutations of the folliculin (FLCN) gene. Chronic obstructive pulmonary disease (COPD), characterised by an irreversible airflow limitation, is primarily caused by cigarette smoking. OBJECTIVE: Given that COPD often shares structural features with BHD, we investigated the link between COPD, cigarette smoke (CS) exposure and FLCN expression. METHODS: We measured the expression of FLCN in human COPD lungs and CS-exposed mouse lungs, as well as in CS extract (CSE)-exposed immortalised human airway epithelial cells by immunoblotting. RESULTS: We found that the lung FLCN protein levels in smokers with COPD and CS exposure mice exhibit a marked decrease compared with smokers without COPD and room air exposure mice, respectively. We confirmed CS induced degradation of FLCN in immortalised human bronchial epithelial Beas-2B cells via ubiquitin proteasome system. Further, siRNA targeting FLCN enhanced CSE-induced cytotoxicity. By contrast, FLCN overexpression protected cells from CSE-induced cytotoxicity. We found that FBXO23, the ubiquitin E3 ligase subunit, specifically binds to and targets FLCN for degradation. Inhibition of ATM (ataxia-telangiectasia mutated) attenuated CSE induced FLCN degradation, suggesting a role of ATM in FLCN proteolysis. We further confirmed that the mutant of major FLCN phosphorylation site serine 62A is resistant to CSE-induced degradation and cytotoxicity. CONCLUSIONS: Our study demonstrates that CS exposure is a secondary cause of FLCN deficiency due to the enhanced proteolysis, which promoted airway epithelial cell death.
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Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Animales , Humanos , Ratones , Fumar Cigarrillos/efectos adversos , Pulmón/química , Pulmón/metabolismo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinas/metabolismoRESUMEN
BACKGROUND: In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. METHODS: We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. RESULTS: TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. CONCLUSIONS: Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.
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Fibrosis Pulmonar Idiopática , Miofibroblastos , Humanos , Ratones , Animales , Miofibroblastos/metabolismo , Cromatina , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Fibroblastos/metabolismo , Colágeno/genética , Colágeno/metabolismo , Fibrosis , Bleomicina , Factores de Transcripción/genética , ARN/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
An increasing number of translational investigations of lung biology rely on analyzing single cell suspensions obtained from human lungs. To obtain these single cell suspensions, human lungs from biopsies or research-consented organ donors must be subjected to mechanical and enzymatic digestion prior to analysis with either flow cytometry or single cell RNA sequencing. A variety of enzymes have been used to perform tissue digestion, each with potential limitations. To better understand the limitations of each enzymatic digestion protocol and to establish a framework for comparing studies across protocols, we performed five commonly published protocols in parallel from identical samples obtained from 6 human lungs. Following mechanical (gentleMACS™) and enzymatic digestion, we quantified cell count and viability using a Nexcelom Cellometer and determined cell phenotype using multiparameter spectral flow cytometry (Cytek™ Aurora). We found that all protocols were superior in cellular yield and viability when compared to mechanical digestion alone. Protocols high in dispase cleaved immune markers CD4, CD8, CD69, and CD103 and contributed to an increased monocyte to macrophage yield. Similarly, dispase led to a differential epithelial cell yield, with increased TSPN8+ and ITGA6+ epithelial cells and reduced CD66e+ cells. When compared to collagenase D, collagenase P protocols yielded increased AT1 and AT2 cells and decreased endothelial cells. These results provide a framework for selecting an enzymatic digestion protocol best suited to the scientific question and allow for comparison of studies using different protocols.
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Colagenasas , Células Endoteliales , Humanos , Citometría de Flujo/métodos , Pulmón , DigestiónRESUMEN
Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.
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COVID-19 , Linfopenia , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Citocinas , Humanos , Infliximab , Leucocitos Mononucleares , Receptores del Factor de Necrosis Tumoral , SARS-CoV-2 , Inhibidores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfaRESUMEN
The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10â d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.
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Cardiomegalia/patología , Sistema de Señalización de MAP Quinasas/fisiología , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas B-raf/fisiología , Animales , Carbamatos/farmacología , Carbamatos/toxicidad , Cardiomegalia/metabolismo , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Dimerización , Técnicas de Sustitución del Gen , Insuficiencia Cardíaca/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Mutación Puntual , Conformación Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/biosíntesis , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Sulfonamidas/toxicidadRESUMEN
Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of systemic sclerosis-associated interstitial lung disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-like receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several damage-associated molecular pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform fibronectin-extra domain A (FN-EDA) are prominent in pathological extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.
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Fibronectinas , Enfermedades Pulmonares Intersticiales , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Ligandos , Enfermedades Pulmonares Intersticiales/metabolismo , Isoformas de Proteínas/metabolismo , Receptor Toll-Like 9/genética , UbiquitinaciónRESUMEN
Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, but the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we describe a significant difference in GCN5L1 levels between male and female mouse hearts, and in the hearts of women between post- and premenopausal age. We further find that estrogen drives GCN5L1 expression in a cardiac cell line and uses pharmacological approaches to determine the mechanism to be G protein-coupled estrogen receptor (GPER) activation, via translational regulation.NEW & NOTEWORTHY We demonstrate here for the first time that mitochondrial protein acetylation is increased in female hearts, associated with an increase in GCN5L1 levels through a GPER-dependent mechanism. These findings reveal a new potential mediator of divergent cardiac mitochondrial function between men and women.
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Proteínas del Tejido Nervioso , Sirtuina 3 , Acetilación , Animales , Estrógenos , Femenino , Corazón/fisiología , Humanos , Masculino , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismoRESUMEN
Rationale: Tissue-resident memory T cells (TRM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (MLR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between MLR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: TRM and MLR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3+ and CD68+ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung TRM and MLR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8+ TRM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that MLR provide costimulatory signaling to PD1hi CD4+ and CD8+ lung TRM,, augmenting the effector cytokine production and degranulation of TRM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human MLR colocalize with and provide costimulation signaling to TRM, augmenting their effector function.
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Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Memoria Inmunológica/fisiología , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/fisiología , Adulto , Técnicas de Cocultivo , Citocinas/metabolismo , Femenino , Humanos , Trasplante de Pulmón , Masculino , Persona de Mediana Edad , Perfusión , Técnicas de Cultivo de TejidosRESUMEN
Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged ß-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute ß-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression (P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.
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Agonistas de Receptores Adrenérgicos beta 2/farmacología , Síndrome de Superposición de la Enfermedad Pulmonar Obstructiva Crónica-Asmática/tratamiento farmacológico , Síndrome de Superposición de la Enfermedad Pulmonar Obstructiva Crónica-Asmática/patología , Broncodilatadores/farmacología , AMP Cíclico/biosíntesis , Macrófagos Alveolares/inmunología , Líquido del Lavado Bronquioalveolar/química , Línea Celular , AMP Cíclico/genética , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Humanos , Células Asesinas Naturales/inmunología , Pulmón/patología , Macrófagos Alveolares/metabolismo , Análisis de la Célula Individual , Células THP-1RESUMEN
BACKGROUND: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown. METHODS: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene. FINDINGS: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death. INTERPRETATION: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.
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Fibrosis Pulmonar Idiopática/microbiología , Trasplante de Pulmón , Pulmón/microbiología , Microbiota/fisiología , Tomografía Computarizada por Rayos X/métodos , Adulto , Anciano , Líquido del Lavado Bronquioalveolar/microbiología , Estudios de Casos y Controles , Progresión de la Enfermedad , Humanos , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/cirugía , Pulmón/diagnóstico por imagen , Pulmón/cirugía , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary disease characterized by increased vascular cell proliferation with apoptosis resistance and occlusive remodeling of the small pulmonary arteries. The Notch family of proteins subserves proximal signaling of an evolutionarily conserved pathway that effects cell proliferation, fate determination, and development. In endothelial cells (ECs), Notch receptor 2 (Notch2) was shown to promote endothelial apoptosis. However, a pro- or antiproliferative role for Notch2 in pulmonary endothelial proliferation and ensuing PAH is unknown. We postulated that suppressed Notch2 signaling drives pulmonary endothelial proliferation in the context of PAH. We observed that levels of Notch2 are ablated in lungs from PAH subjects compared with non-PAH controls. Notch2 expression was attenuated in human pulmonary artery endothelial cells (hPAECs) exposed to vasoactive stimuli including hypoxia, TGF-ß, ET-1, and IGF-1. Notch2-deficient hPAECs activated Akt, Erk1/2, and antiapoptotic protein Bcl-2 and reduced levels of p21cip and Bax associated with increased EC proliferation and reduced apoptosis. In addition, Notch2 suppression elicited a paradoxical activation of Notch1 and canonical Notch target gene Hes1, Hey1, and Hey2 transcription. Furthermore, reduction in Rb and increased E2F1 binding to the Notch1 promoter appear to explain the Notch1 upregulation. Yet, when Notch1 was decreased in Notch2-suppressed cells, the wound injury response was augmented. In aggregate, our results demonstrate that loss of Notch2 in hPAECs derepresses Notch1 and elicits EC hallmarks of PAH. Augmented EC proliferation upon Notch1 knockdown points to a context-dependent role for Notch1 and 2 in endothelial cell homeostasis.NEW & NOTEWORTHY This study demonstrates a previously unidentified role for Notch2 in the maintenance of lung vascular endothelial cell quiescence and pulmonary artery hypertension (PAH). A key novel finding is that Notch2 suppression activates Notch1 via Rb-E2F1-mediated signaling and induces proliferation and apoptosis resistance in human pulmonary artery endothelial cells. Notably, PAH patients show reduced levels of endothelial Notch2 in their pulmonary arteries, supporting Notch2 as a fundamental driver of PAH pathogenesis.
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Proliferación Celular , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Receptor Notch2/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Células Endoteliales/fisiología , Endotelio Vascular/citología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/genética , Proteínas Represoras/metabolismo , Factor de Transcripción HES-1/metabolismoRESUMEN
BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.