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Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease characterized by fibrosis. Two FDA-approved drugs, pirfenidone and nintedanib, only modestly prolong survival. In this study, we asked whether levels of select circulating biomarkers in patients with IPF demonstrated changes in response to treatment over time and whether treatment with pirfenidone and nintedanib led to differential biomarker expression. Serial plasma samples from 48 patients with IPF on usual treatment and six healthy volunteers were analyzed to identify differentially expressed blood protein. Hypothesis-driven potential biomarker selection was based on recent literature, internal preclinical data, and the PROLIFIC Consortium (Schafer P. 6th Annual IPF Summit. Boston, MA, 2022) proposed biomarkers of pulmonary fibrosis. We compared our findings to public databases to provide insights into relevant signaling pathways in IPF. Of the 26 proteins measured, we found that 11 (SP-D, TIMP1, MMP7, CYFRA21-1, YKL40, CA125, sICAM, IP-10, MDC, CXCL13) were significantly elevated in patients with IPF compared with healthy volunteers but their levels did not significantly change over time. In the IPF samples, seven proteins were elevated in the treatment group compared with the no-treatment group. However, protein profiles were not distinguishable between patients on pirfenidone versus nintedanib. We demonstrated that most proteins differentially detected in our samples were predicted to be secreted from the lung epithelial or interstitial compartments. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. Understanding the contributions of the systemic response in IPF may be important as new therapeutics are developed.NEW & NOTEWORTHY In this study, we confirmed protein expression differences in only a subset of predicted biomarkers from IPF and control subjects. Most differentially expressed proteins were predicted to be secreted from lung cells. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. The contributions of the systemic response in IPF may be important as new therapeutics are developed.
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Antígenos de Neoplasias , Fibrosis Pulmonar Idiopática , Queratina-19 , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Fibrosis , BiomarcadoresRESUMEN
Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8ß1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed Npnt allele were used to generate global knockouts (KOs). Staged embryos were obtained by timed matings of heterozygotes and lungs were isolated for analysis. Although primary and secondary lung bud formation was normal in KO embryos, fusion of right lung lobes, primarily the medial and caudal, was first detected at E13.5 and persisted into adulthood. The lung parenchyma of KO mice was indistinguishable from wild-type (WT) and lobe fusion did not alter respiratory mechanics in adult KO mice. Interrogation of an existing single-cell RNA-seq atlas of embryonic and adult mouse lungs identified Npnt transcripts in mesothelial cells at E12.5 and into the early postnatal period, but not in adult lungs. KO embryonic lungs exhibited increased expression of laminin α5 and deposition of collagen IV in the mesothelial BM, accompanied by abnormalities in collagen fibrils in the adjacent stroma. Cranial and accessory lobes extracted from KO embryonic lungs fused ex vivo when cultured in juxtaposition, with the area of fusion showing loss of the mesothelial marker Wilms tumor 1. Because a similar pattern of lobe fusion was previously observed in integrin α8 KO embryos, our results suggest that NPNT signaling through integrin α8, likely in the visceral pleura, maintains right lung lobe separation during embryogenesis.
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Proteínas de la Matriz Extracelular , Proteínas de la Membrana , Animales , Ratones , Proteínas de la Matriz Extracelular/genética , Desarrollo Embrionario/genética , Pulmón/metabolismo , ColágenoRESUMEN
We previously showed that pericyte-like cells derived from the FoxD1-lineage contribute to myofibroblasts following bleomycin-induced lung injury. However, their functional significance in lung fibrosis remains unknown. In this study, we used a model of lung pericyte-like cell ablation to test the hypothesis that pericyte-like cell ablation attenuates lung fibrosis in bleomycin-induced lung injury. Lung fibrosis was induced by intratracheal instillation of bleomycin. To ablate pericyte-like cells in the lung, diphtheria toxin (DT) was administered to Foxd1-Cre;Rosa26-iDTR mice at two different phases of bleomycin-induced lung injury. For early ablation, we coadministered bleomycin with DT and harvested mice at days 7 and 21. To test the effect of ablation after acute injury, we delivered DT 7 days after bleomycin administration. We assessed fibrosis by lung hydroxyproline content and semiquantitative analysis of picrosirius red staining. We performed bronchoalveolar lavage to determine cell count and differential. We also interrogated mRNA expression of fibrosis-related genes in whole lung RNA. Compared with DT-insensitive littermates where pericyte-like cells were not ablated, DT-sensitive animals exhibited no difference in fibrosis at day 21 both in the early and late pericyte ablation models. However, early ablation of pericytes reduced acute lung inflammation, as indicated by decreased inflammatory cells. Our data confirm a role for pericytes in regulating pulmonary inflammation in early lung injury.
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Lesión Pulmonar , Fibrosis Pulmonar , Animales , Bleomicina/farmacología , Líquido del Lavado Bronquioalveolar , Hidroxiprolina , Pulmón/metabolismo , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/patología , Lesión Pulmonar/terapia , Ratones , Ratones Endogámicos C57BL , Pericitos/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
Pericytes apposed to the capillary endothelium are known to stabilize and promote endothelial integrity. Recent studies indicate that lung pericytes play a prominent role in lung physiology, and they are involved in the development of various lung diseases including lung injury in sepsis, pulmonary fibrosis, asthma, and pulmonary hypertension. Accordingly, human lung pericyte studies are important for understanding the mechanistic basis of lung physiology and pathophysiology; however, human lung pericytes can only be cultured for a few passages and no immortalized human lung pericyte cell line has been established so far. Thus, our study aims to establish an immortalized human lung pericyte cell line. Developed using SV40 large T antigen lentivirus, immortalized pericytes exhibit stable SV40T expression, sustained proliferation, and have significantly higher telomerase activity compared to normal human lung pericytes. In addition, these cells retained pericyte characteristics, marked by similar morphology, and expression of pericyte cell surface markers such as PDGFRß, NG2, CD44, CD146, CD90, and CD73. Furthermore, similar to that of primary pericytes, immortalized pericytes promoted endothelial cell tube formation and responded to different stimuli. Our previous data showed that friend leukemia virus integration 1 (Fli-1), a member of the ETS transcription factor family, is a key regulator that modulates inflammatory responses in mouse lung pericytes. We further demonstrated that Fli-1 regulates inflammatory responses in immortalized human lung pericytes. To summarize, we successfully established an immortalized human lung pericyte cell line, which serves as a promising tool for in vitro pericyte studies to understand human lung pericyte physiology and pathophysiology.
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Línea Celular , Pulmón/citología , Pericitos , Proteínas de Microfilamentos/metabolismo , Transactivadores/metabolismoRESUMEN
Antiretroviral therapy in HIV patients has lengthened lifespan but led to an increased risk for secondary comorbidities, such as pulmonary complications characterized by vascular dysfunction. In the lung, PDGFRß+ mesenchymal cells known as pericytes intimately associate with endothelial cells and are key for their survival both structurally and through the secretion of prosurvival factors. We hypothesize that in HIV infection there are functional changes in pericytes that may lead to destabilization of the microvasculature and ultimately to pulmonary abnormalities. Our objective in this study was to determine whether lung pericytes could be directly infected with HIV. We leveraged lung samples from macaque lungs with or without SIV infection and normal human lung for in vitro experiments. Pericytes were isolated based on the marker platelet-derived growth factor receptor-ß (PDGFRß). We determined that lung PDGFRß-positive (PDGFRß+) pericytes from both macaques and humans express CD4, the primary receptor for SIV/HIV, as well as the major coreceptors CXCR4 and CCR5. We found cells positive for both PDGFRß and SIV in lungs from infected macaques. Lung pericytes isolated from these animals also harbored detectable SIV. To confirm relevance to human disease, we demonstrated that human lung pericytes are capable of being productively infected by HIV in vitro, with the time course of infection suggesting development of viral latency. In summary, we show for the first time that SIV/HIV directly infects lung pericytes, implicating these cells as a novel target and potential reservoir for the virus in vivo.
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Células Endoteliales/virología , Infecciones por VIH/virología , Pulmón/virología , Macrófagos/virología , Linfocitos T CD4-Positivos/virología , Humanos , Pulmón/inmunología , Macrófagos/inmunología , Receptores CXCR4/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Latencia del Virus/fisiología , Replicación ViralRESUMEN
The lung has numerous roles, including gas exchange, immune surveillance, and barrier function. Being a highly vascularized organ, the lung receives dual blood supply from both the pulmonary and bronchial circulation. Therefore, pericytes likely play a prominent role in lung physiology given their localization in the perivascular niche. New genetic approaches have increased our understanding of the origin and the diverse functions of lung pericytes. Lung pericytes are myofibroblast progenitors, contributing to development of fibrosis in mouse models. Lung pericytes are also capable of responding to danger signals and amplify the inflammatory response through elaboration of cytokines and adhesion molecules. In this chapter, we describe the molecular, anatomical, and phenotypical characterization of lung pericytes. We further highlight their potential roles in the pathogenesis of lung diseases including pulmonary fibrosis, asthma, and pulmonary hypertension. Finally, current gaps in knowledge and areas of ongoing investigation in lung pericyte biology are also discussed.
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Pulmón/citología , Miofibroblastos/citología , Pericitos/citología , Animales , Asma , Humanos , Hipertensión Pulmonar , Ratones , Fibrosis PulmonarRESUMEN
Background: Pericytes are vascular mural cells and are embedded in the basement membrane of the microvasculature. Recent studies suggest a role for pericytes in lipopolysaccharide (LPS)-induced microvascular dysfunction and mortality, but the mechanisms of pericyte loss in sepsis are largely unknown. Methods: By using a cecal ligation and puncture (CLP)-induced murine model of sepsis, we observed that CLP led to lung and renal pericyte loss and reduced lung pericyte density and pericyte/endothelial cell (EC) coverage. Results: Up-regulated Friend leukemia virus integration 1 (Fli-1) messenger ribonucleic acid (RNA) and protein levels were found in lung pericytes from CLP mice in vivo and in LPS-stimulated lung pericytes in vitro. Knockout of Fli-1 in Foxd1-derived pericytes prevented CLP-induced pericyte loss, vascular leak, and improved survival. Disrupted Fli-1 expression by small interfering RNA inhibited LPS-induced inflammatory cytokines and chemokines in cultured lung pericytes. Furthermore, CLP-induced pericyte pyroptosis was mitigated in pericyte Fli-1 knockout mice. Conclusions: Our findings suggest that Fli-1 is a potential therapeutic target in sepsis.
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Pericitos/fisiología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Sepsis/metabolismo , Animales , Ciego , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inflamación/metabolismo , Ligadura , Lipopolisacáridos , Pulmón/citología , Ratones , Ratones Noqueados , Piroptosis , Sepsis/inmunología , Regulación hacia ArribaRESUMEN
Pericytes are key regulators of the microvasculature through their close interactions with the endothelium. However, pericytes play additional roles in tissue homeostasis and repair, in part by transitioning into myofibroblasts. Accumulation of myofibroblasts is a hallmark of fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). To understand the contribution and role of pericytes in human lung fibrosis, we isolated these cells from non-IPF control and IPF lung tissues based on expression of platelet-derived growth factor receptor-ß (PDGFR-ß), a common marker of pericytes. When cultured in a specialized growth medium, PDGFR-ß+ cells retain the morphology and marker profile typical of pericytes. We found that IPF pericytes migrated more rapidly and invaded a basement membrane matrix more readily than control pericytes. Exposure of cells to transforming growth factor-ß, a major fibrosis-inducing cytokine, increased expression of α-smooth muscle actin and extracellular matrix genes in both control and IPF pericytes. Given that pericytes are uniquely positioned in vivo to respond to danger signals of both systemic and tissue origin, we stimulated human lung pericytes with agonists having pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Both control and IPF lung pericytes increased expression of proinflammatory chemokines in response to specific PAMPs and DAMPs released from necrotic cells. Our results suggest that control and IPF lung pericytes are poised to react to tissue damage, as well as microbial and fibrotic stimuli. However, IPF pericytes are primed for migration and matrix invasion, features that may contribute to the function of these cells in lung fibrosis.
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Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Pulmón/patología , Pericitos/metabolismo , Pericitos/patología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Adulto , Anciano , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Masculino , Persona de Mediana Edad , Miofibroblastos/metabolismo , Miofibroblastos/patología , Factor de Crecimiento Transformador beta/metabolismo , Adulto JovenRESUMEN
Emphysema is one of the most common lung diseases in HIV+ individuals. The pathogenesis of HIV-associated emphysema remains unclear; however, radiographic distribution and earlier age of presentation of emphysema in the lungs of HIV+ patients are similar to deficiency of α1-antitrypsin (A1AT), a key elastase inhibitor in the lung. Reduced levels of circulating A1AT in HIV+ patients suggest a potential mechanism for emphysema development. In the present study we asked if A1AT levels and activity in the bronchoalveolar lavage fluid (BALF) differ in HIV+ and HIV- patients with and without emphysema. A1AT levels were measured by ELISA in plasma and BALF from a cohort of 21 HIV+ and 29 HIV- patients with or without emphysematous changes on chest CT scan. To analyze A1AT function, we measured elastase activity in the BALF and assessed oxidation and polymerization of A1AT by Western blotting. Total A1AT was increased in the BALF, but not in the plasma, of HIV+ compared with HIV- patients, regardless of the presence or absence of emphysema. However, antielastase activity was decreased in BALF from HIV+ patients, suggesting impaired A1AT function. Higher levels of the oxidized form of A1AT were detected in BALF from HIV+ than HIV- patients, which may account for the decreased antielastase activity. These findings suggest that, in the lungs of HIV+ patients, posttranslational modifications of A1AT produce a "functional deficiency" of this critical elastase inhibitor, which may contribute to emphysema development.
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Biomarcadores/sangre , Infecciones por VIH/complicaciones , Enfisema Pulmonar/sangre , Inhibidores de Tripsina/sangre , alfa 1-Antitripsina/sangre , Líquido del Lavado Bronquioalveolar , Estudios de Cohortes , Femenino , VIH/aislamiento & purificación , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana Edad , Enfisema Pulmonar/etiología , Enfisema Pulmonar/patologíaRESUMEN
RATIONALE: Apoptotic cell phagocytosis (efferocytosis) is mediated by specific receptors and is essential for resolution of inflammation. In chronic inflammation, apoptotic cell clearance is dysfunctional and soluble levels of several apoptotic cell receptors are elevated. Reports have identified proteolytic cleavage as a mechanism capable of releasing soluble apoptotic cell receptors, but the functional implications of their proteolysis are unclear. OBJECTIVE: To test the hypothesis that ADAM17-mediated cleavage of apoptotic cell receptors limits efferocytosis in vivo. METHODS AND RESULTS: In vivo comparison of macrophage efferocytosis in wild-type and Adam17-null hematopoietic chimeras demonstrates that ADAM17 deficiency leads to a 60% increase in efferocytosis and an enhanced anti-inflammatory phenotype in a model of peritonitis. In vitro uptake of phosphatidylserine liposomes identifies the dual-pass apoptotic cell receptor CD36 as a major contributor to enhanced efferocytosis, and CD36 surface levels are elevated on macrophages from Adam17-null mice. Further, temporal elevation of CD36 expression with inflammation may also contribute to its impact. Soluble CD36 from macrophage-conditioned media comprises 2 species based on Western blotting, and mass spectrometry identifies 3 N-terminal peptides that represent probable cleavage sites. Levels of soluble CD36 are decreased in Adam17-null conditioned media, providing evidence for involvement of ADAM17 in CD36 cleavage. Importantly, enhanced efferocytosis in vivo by macrophages lacking ADAM17 is CD36 dependent and accelerates macrophage clearance from the peritoneum, thus promoting resolution of inflammation and highlighting the impact of increased apoptotic cell uptake. CONCLUSIONS: Our studies demonstrate the importance of ADAM17-mediated proteolysis for in vivo efferocytosis regulation and suggest a possible mechanistic link between chronic inflammation and defective efferocytosis.
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Proteínas ADAM/fisiología , Apoptosis/fisiología , Antígenos CD36/fisiología , Macrófagos Peritoneales/enzimología , Peritonitis/enzimología , Fagocitosis/fisiología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Trasplante de Médula Ósea , Antígenos CD36/química , Quimera , Medios de Cultivo Condicionados/farmacología , Dexametasona/farmacología , Células Madre Embrionarias/trasplante , Liposomas , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Peritonitis/inducido químicamente , Peritonitis/patología , Fenotipo , Tioglicolatos/toxicidad , Timocitos/patología , Timocitos/trasplanteAsunto(s)
Fibrosis Pulmonar Idiopática , Animales , Bleomicina , Ratones , Proteínas Tirosina Quinasas , Quinolinas , Tiourea/análogos & derivados , ArmasRESUMEN
p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17(-/-) MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17(-/-) MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.
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Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Probióticos/metabolismo , Activación Transcripcional , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Línea Celular Tumoral , Activación Enzimática/genética , Células Epiteliales/citología , Células Epiteliales/microbiología , Receptores ErbB/genética , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Among the pleotropic effects of endothelial nitric oxide (NO) is protection against vascular inflammation during high-fat diet (HFD) feeding. The current work investigated the role of the enzyme vasodilatory-stimulated phosphoprotein (VASP) as a downstream mediator of the anti-inflammatory effect of NO signaling in vascular tissue. Relative to mice fed a low-fat diet (LFD), levels of VASP Ser(239) phosphorylation, a marker of VASP activation, were dramatically reduced in aortic tissue of mice with obesity induced by consuming a HFD. As reported previously, the effect of the HFD was associated with increased aortic inflammation, as measured by increased NF-κB-dependent gene expression, and reduced vascular insulin sensitivity (including insulin-stimulated phosphorylation of eNOS and Akt). These effects of the HFD were recapitulated by VASP knockout, implying a physiological role for VASP to constrain inflammatory signaling and thereby maintain vascular insulin sensitivity. Conversely, overexpression of VASP in endothelial cells blocked inflammation and insulin resistance induced by palmitate. The finding that transplantation of bone marrow from VASP-deficient donors into normal recipients does not recapitulate the vascular effects of whole body VASP deficiency suggests that the protective effects of this enzyme are not mediated in immune or other bone marrow-derived cells. These studies implicate VASP as a downstream mediator of the NO/cGMP pathway that is both necessary and sufficient to protect against vascular inflammation and insulin resistance. As such, this work identifies VASP as a potential therapeutic target in the treatment of obesity-related vascular dysfunction.
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Aorta/metabolismo , Moléculas de Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Resistencia a la Insulina , Proteínas de Microfilamentos/fisiología , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Fosfoproteínas/fisiología , Vasculitis/metabolismo , Animales , Aorta/citología , Aorta/inmunología , Trasplante de Médula Ósea , Bovinos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Dieta Alta en Grasa , Células Endoteliales/inmunología , Perfilación de la Expresión Génica , Humanos , Inflamación/metabolismo , Resistencia a la Insulina/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microvasos/citología , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo III/genética , Obesidad/inmunología , Palmitatos/farmacología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Vasculitis/inmunologíaRESUMEN
Systemic sclerosis (SSc) is a chronic disease characterized by fibrosis and vascular abnormalities in the skin and internal organs, including the lung. SSc-associated pulmonary fibrosis (SSc-PF) is the leading cause of death in SSc patients. Pericytes are key regulators of vascular integrity and endothelial function. The role that pericytes play in SSc-PF remains unclear. We compared the transcriptome of pericytes from SSc-PF lungs (SScL) to pericytes from normal lungs (NORML). We identified 1,179 differentially expressed genes in SScL pericytes. Pathways enriched in SScL pericytes included prostaglandin, PI3K-AKT, calcium, and vascular remodeling signaling. Decreased cyclic AMP production and altered phosphorylation of AKT in response to prostaglandin E2 in SScL pericytes demonstrate the functional consequence of changes in the prostaglandin pathway that may contribute to fibrosis. The transcriptomic signature of SSc lung pericytes suggests that they promote vascular dysfunction and contribute to the loss of protection against lung inflammation and fibrosis.
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Endothelial dysfunction is a critical feature of acute respiratory distress syndrome (ARDS) associated with higher disease severity and worse outcomes. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses and heterogeneity of human host responses. Use of microphysiological systems (MPS) to investigate lung endothelial function may shed light on underlying mechanisms and targeted treatments for ARDS. We assessed the response to plasma from critically ill sepsis patients in our lung endothelial MPS through measurement of endothelial permeability, expression of adhesion molecules, and inflammatory cytokine secretion. Sepsis plasma induced areas of endothelial cell (EC) contraction, loss of cellular coverage, and luminal defects. EC barrier function was significantly worse following incubation with sepsis plasma compared to healthy plasma. EC ICAM-1 expression, IL-6 and soluble ICAM-1 secretion increased significantly more after incubation with sepsis plasma compared with healthy plasma. Plasma from sepsis patients who developed ARDS further increased IL-6 and sICAM-1 compared to plasma from sepsis patients without ARDS and healthy plasma. Our results demonstrate the proof of concept that lung endothelial MPS can enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients.
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Células Endoteliales , Pulmón , Síndrome de Dificultad Respiratoria , Sepsis , Humanos , Células Endoteliales/metabolismo , Molécula 1 de Adhesión Intercelular/sangre , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-6/sangre , Interleucina-6/metabolismo , Pulmón/fisiopatología , Pulmón/metabolismo , Sistemas Microfisiológicos , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria/metabolismo , Sepsis/fisiopatología , Sepsis/complicaciones , Sepsis/metabolismoRESUMEN
Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin ß2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin ß2 from mouse macrophages are demonstrated. Soluble integrin ß2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin ß2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin ß2 is accelerated, and antibody disruption of integrin ß2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin ß2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.
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Antígenos CD18/metabolismo , Movimiento Celular , Macrófagos Peritoneales/metabolismo , Metaloproteasas/metabolismo , Peritonitis/metabolismo , Multimerización de Proteína , Animales , Antígenos CD18/genética , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Fibrina/genética , Fibrina/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Macrófagos Peritoneales/patología , Metaloproteasas/genética , Ratones , Ratones Mutantes , Peritonitis/genética , Peritonitis/patología , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismoRESUMEN
Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to α(v) -containing integrins, and the SLAYGLR sequence binding to α(4) ß(1), α(4) ß(7), and α(9) ß(1) integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated-bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α(4) integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α(4) and α(9) integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL-12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid-derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate-elicited peritonitis. Collectively, these data indicate that α(4) and α(9) integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation.
Asunto(s)
Quimiotaxis , Cadenas alfa de Integrinas/metabolismo , Integrina alfa4/metabolismo , Macrófagos/citología , Osteopontina/metabolismo , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/citología , Supervivencia Celular , Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Osteopontina/química , Peritonitis/metabolismo , Peritonitis/patología , Fenotipo , Estructura Terciaria de Proteína , Transducción de Señal , Relación Estructura-Actividad , TioglicolatosRESUMEN
TNF-α-converting enzyme (TACE, herein denoted as Adam17) proteolytically sheds several cell-surface inflammatory proteins, but the physiologic importance of the cleavage of these substrates from leukocyte subsets during inflammation is incompletely understood. In this study, we show that Adam17-null neutrophils have a 2-fold advantage in their initial recruitment during thioglycollate-induced peritonitis, and they roll slower and adhere more readily in the cremaster model than wild-type neutrophils. Although CD44 and ICAM-1 are both in vitro substrates of Adam17, their surface levels are not altered on Adam17-null neutrophils. In contrast, L-selectin levels are elevated up to 10-fold in Adam17-null circulating neutrophils, and their accelerated peritoneal influx, slower rolling, and increased adhesion in the cremaster muscle are dependent on L-selectin. Analysis of mixed chimeras shows that enhanced L-selectin levels and accelerated influx were both cell-intrinsic properties of neutrophils lacking Adam17. In contrast, Adam17-null monocytes display no acceleration of infiltration into the peritoneum in spite of elevated L-selectin surface levels, and their peritoneal influx was independent of L-selectin. Therefore, our data demonstrate substrate and myeloid cell-type specificity of Adam17-mediated cleavage of its substrates, and show that neutrophils and monocytes use distinct mechanisms for infiltration of tissues.
Asunto(s)
Proteínas ADAM/inmunología , Proteínas ADAM/metabolismo , Movimiento Celular/inmunología , Inflamación/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Adhesión Celular/inmunología , Quimera , Modelos Animales de Enfermedad , Fragmentos Fab de Inmunoglobulinas/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Selectina L/inmunología , Selectina L/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Monocitos/citología , Neutrófilos/citología , Peritonitis/inducido químicamente , Peritonitis/inmunología , Peritonitis/metabolismo , Especificidad por Sustrato , Tioglicolatos/farmacologíaRESUMEN
Acute respiratory distress syndrome due to non-pulmonary causes exhibits prominent endothelial activation which is challenging to assess in critically ill patients. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses. Use of microphysiological systems (MPS) offer improved fidelity to human biological responses and better predict pharmacological responses than traditional culture. We adapted a lung endothelial MPS based on the LumeNEXT platform to evaluate the effect of plasma from critically ill sepsis patients on endothelial permeability, adhesion molecule expression and inflammatory cytokine production. Lumens incubated with sepsis plasma exhibited areas of contraction, loss of cellular coverage, and luminal defects. Sepsis plasma-incubated lumens had significantly increased permeability compared to lumens incubated with healthy donor plasma. ICAM-1 expression increased significantly in lumens incubated with sepsis plasma compared with those incubated with healthy control plasma, while concentrations of IL-6, IL-18, and soluble VEGF-R1 increased in sepsis plasma before and after incubation in the MPS compared with healthy control plasma. Use of the lung endothelial MPS may enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients.
RESUMEN
Acute injury of the lung involves damage to the epithelium and its underlying extracellular matrix (ECM), the basement membrane (BM). How BMs contribute to injury resolution is poorly understood. Nephronectin (NPNT) is a high-affinity ligand for integrin α8ß1 and, although first identified in the mouse kidney, is prominently expressed in the lung, where it localizes to BMs in the alveoli. To determine if NPNT plays a role in acute injury and inflammation of the lung, we developed a model for postnatal deletion of NPNT using mice with a floxed allele of Npnt in combination with a tamoxifen-inducible Cre recombinase expressed at the ROSA locus. Expression of NPNT was substantially reduced in lungs from tamoxifen-treated Cre+ animals. Cre+ mice and Cre- controls were given E. coli LPS by oropharyngeal aspiration to induce injury and inflammation. In Cre- lungs, although both Npnt and Itga8 (integrin α8) transcripts were downregulated at the peak of inflammation, NPNT protein was still detectable. While the onset of inflammation was similar for Cre+ and Cre-, NPNT-deficient lungs still had thickened alveolar septa and there were increased macrophages in the bronchoalveolar lavage fluid (BALF) in the resolution phase. BALF from Cre+ lungs was more chemotactic for bone marrow-derived macrophages than Cre- in in vitro experiments, but there were no differences in the elaboration of chemokines in vivo. We speculate that absence of NPNT in BMs of the alveoli impairs or delays inflammatory and injury resolution in this model, but further studies are needed to establish the precise role of NPNT in tissue repair.