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OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.
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Antígenos de Superficie , Enfermedad de Crohn , Matriz Extracelular , Fibrosis , Proteínas de la Leche , Humanos , Animales , Enfermedad de Crohn/patología , Enfermedad de Crohn/metabolismo , Proteínas de la Leche/metabolismo , Proteínas de la Leche/farmacología , Antígenos de Superficie/metabolismo , Matriz Extracelular/metabolismo , Miofibroblastos/metabolismo , Modelos Animales de Enfermedad , Ratones , RatasRESUMEN
Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.
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Fibrosis Pulmonar Idiopática , Mecanotransducción Celular , Miofibroblastos , Proteína de Unión al Calcio S100A4 , Animales , Ratones , Transdiferenciación Celular , Fibrosis , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismoRESUMEN
BACKGROUND: Systemic sclerosis (SSc) is a rare, complex, connective tissue disorder. Interstitial lung disease (ILD) is common in SSc, occurring in 35-52% of patients and accounting for 20-40% of mortality. Evolution of therapeutic options has resulted in a lack of consensus on how to manage this condition. This Delphi study was initiated to develop consensus recommendations based on expert physician insights regarding screening, progression, treatment criteria, monitoring of response, and the role of recent therapeutic advances with antifibrotics and immunosuppressants in patients with SSc-ILD. METHODS: A modified Delphi process was completed by pulmonologists (n = 13) and rheumatologists (n = 12) with expertise in the management of patients with SSc-ILD. Panelists rated their agreement with each statement on a Likert scale from - 5 (complete disagreement) to + 5 (complete agreement). Consensus was predefined as a mean Likert scale score of ≤ - 2.5 or ≥ + 2.5 with a standard deviation not crossing zero. RESULTS: Panelists recommended that all patients with SSc be screened for ILD by chest auscultation, spirometry with diffusing capacity of the lungs for carbon monoxide, high-resolution computed tomography (HRCT), and/or autoantibody testing. Treatment decisions were influenced by baseline and changes in pulmonary function tests, extent of ILD on HRCT, duration and degree of dyspnea, presence of pulmonary hypertension, and potential contribution of reflux. Treatment success was defined as stabilization or improvement of signs or symptoms of ILD and functional status. Mycophenolate mofetil was identified as the initial treatment of choice. Experts considered nintedanib a therapeutic option in patients with progressive fibrotic ILD despite immunosuppressive therapy or patients contraindicated/unable to tolerate immunotherapy. Concomitant use of nintedanib with MMF/cyclophosphamide can be considered in patients with advanced disease at initial presentation, aggressive ILD, or significant disease progression. Although limited consensus was achieved on the use of tocilizumab, the experts considered it a therapeutic option for patients with early SSc and ILD with elevated acute-phase reactants. CONCLUSIONS: This modified Delphi study generated consensus recommendations for management of patients with SSc-ILD in a real-world setting. Findings from this study provide a management algorithm that will be helpful for treating patients with SSc-ILD and addresses a significant unmet need.
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Enfermedades Pulmonares Intersticiales , Esclerodermia Sistémica , Humanos , Consenso , Inmunosupresores/uso terapéutico , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/tratamiento farmacológico , Enfermedades Pulmonares Intersticiales/etiología , Pulmón , Ácido Micofenólico/uso terapéutico , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/terapiaRESUMEN
RATIONALE: Transbronchial cryobiopsy has been increasingly used to diagnose interstitial lung diseases. However, there is uncertainty regarding its accuracy and risks, mainly due to a paucity of prospective or randomized trials comparing cryobiopsy to surgical biopsy. OBJECTIVES: To evaluate the diagnostic yield and complications of cryobiopsy in patients selected by multidisciplinary discussion. METHODS: This was a prospective cohort from 2017 to 2019. We included consecutive patients with suspected interstitial lung diseases being considered for lung biopsy presented at our multidisciplinary meeting. MEASUREMENTS AND MAIN RESULTS: Of 112 patients, we recommended no biopsy in 31, transbronchial forceps biopsy in 16, cryobiopsy in 54 and surgical biopsy in 11. By the end of the study, 34 patients had had cryobiopsy and 24 patients, surgical biopsy. Overall pathologic and multidisciplinary diagnostic yield of cryobiopsy was 47.1% and 61.8%, respectively. The yield increased over time for both pathologic (year 1: 28.6%, year 2: 54.5%, year 3: 66.7%, p = 0.161) and multidisciplinary (year 1: 50%, year 2: 63.6%, year 3: 77.8%, p = 0.412) diagnosis. Overall rate of grade 4 bleeding after cryobiopsy was 11.8%. Cryobiopsy required less chest tube placement (11.8% vs 100%, p < 0.001) and less hospitalizations compared to surgical biopsy (26.5% vs 95.7%, p < 0.001), but hospitalized patients had a longer median hospital stay (2 days vs 1 day, p = 0.004). CONCLUSIONS: Diagnostic yield of cryobiopsy increased over time but the overall grade 4 bleeding rate was 11.8%.
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Enfermedades Pulmonares Intersticiales , Biopsia/efectos adversos , Hemorragia/etiología , Humanos , Enfermedades Pulmonares Intersticiales/complicaciones , Estudios Prospectivos , Instrumentos Quirúrgicos/efectos adversosRESUMEN
OBJECTIVE: Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN: Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS: Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-ß1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5ß1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION: Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.
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Adipocitos/patología , Movimiento Celular , Enfermedad de Crohn/patología , Intestinos/patología , Músculo Liso/patología , Adipogénesis/fisiología , Tejido Adiposo/patología , Diferenciación Celular , Células Cultivadas , Matriz Extracelular/patología , Fibronectinas/metabolismo , Humanos , Andamios del TejidoRESUMEN
The importance of innate immune cells to sense and respond to their physical environment is becoming increasingly recognized. Innate immune cells (e.g. macrophages and neutrophils) are able to receive mechanical signals through several mechanisms. In this review, we discuss the role of mechanosensitive ion channels, such as Piezo1 and transient receptor potential vanilloid 4 (TRPV4), and cell adhesion molecules, such as integrins, selectins, and cadherins in biology and human disease. Furthermore, we explain that these mechanical stimuli activate intracellular signaling pathways, such as MAPK (p38, JNK), YAP/TAZ, EDN1, NF-kB, and HIF-1α, to induce protein conformation changes and modulate gene expression to drive cellular function. Understanding the mechanisms by which immune cells interpret mechanosensitive information presents potential targets to treat human disease. Important areas of future study in this area include autoimmune, allergic, infectious, and malignant conditions.
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Inmunidad Innata/inmunología , Macrófagos/inmunología , Mecanotransducción Celular/inmunología , Neutrófilos/inmunología , Transducción de Señal/inmunología , Animales , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Canales Iónicos/inmunología , Canales Iónicos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Canales Catiónicos TRPV/inmunología , Canales Catiónicos TRPV/metabolismoRESUMEN
PURPOSE: Nintedanib is an approved treatment for idiopathic pulmonary fibrosis (IPF), which slows disease progression. Management of patients with IPF receiving nintedanib can be complicated by tolerability issues, comorbidities, and concomitant medications. We developed consensus recommendations on the management of dosing, adverse events and comorbidities in patients with IPF treated with nintedanib. METHODS: A modified Delphi process using 3 questionnaires was used to survey 14 pulmonologists experienced in using nintedanib. Panelists rated their agreement with statements on a Likert scale from -5 (strongly disagree) to +5 (strongly agree). Consensus was predefined as a mean score of ⩽-2.5 or ⩾+2.5 with a standard deviation not crossing zero. RESULTS: The panelists' recommendations were largely aligned with clinical trial data, real-world evidence, and the prescribing information, and provided additional guidance regarding minimizing gastrointestinal effects, periodic monitoring for liver dysfunction, caution with respect to concomitant administration of cytochrome P450 3A4 and P-glycoprotein 1 inhibitors and inducers and anticoagulants, and management of comorbidities. The panelists unanimously agreed that adverse event management should be individualized, based on careful consideration of the risks and benefits of each possible intervention and discussion with the patient. CONCLUSIONS: These consensus recommendations provide additional guidance on the appropriate management of IPF with nintedanib, for use alongside evidence-based literature and the prescribing information.
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Ion channels/pumps are essential regulators of innate immune cell function. Macrophages have been increasingly recognized to have phenotypic plasticity and location-specific functions in the lung. Transient receptor potential vanilloid 4 (TRPV4) function in lung injury has been shown to be stimulus- and cell-type specific. In the current review, we discuss the importance of TRPV4 in macrophages and its role in phagocytosis and cytokine secretion in acute lung injury/acute respiratory distress syndrome (ARDS). Furthermore, TRPV4 controls a MAPK molecular switch from predominately c-Jun N-terminal kinase, JNK activation, to that of p38 activation, that mediates phagocytosis and cytokine secretion in a matrix stiffness-dependent manner. Expanding knowledge regarding the downstream mechanisms by which TRPV4 acts to tailor macrophage function in pulmonary inflammatory diseases will allow for formulation of novel therapeutics.
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Susceptibilidad a Enfermedades , Inmunidad Innata , Inmunomodulación , Neumonía/etiología , Neumonía/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Plasticidad de la Célula , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Neumonía/patología , Transducción de Señal , Canales Catiónicos TRPV/genéticaRESUMEN
Analyses of COVID-19 patients with preexisting interstitial lung disease (ILD) or pulmonary sarcoidosis is lacking but registries are ongoing. Treatment of COVID-19 in patients with underlying ILD or sarcoidosis may include hospital admission, possible drug treatment, caution with steroids, and avoidance of mechanical ventilation in acute exacerbation of ILD. Patients with COVID-19 respiratory illness are at risk for developing ILD.
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BACKGROUND: The diagnosis of cardiac sarcoidosis (CS) is challenging. Because of the current limitations of endomyocardial biopsy as a reference standard, physicians rely on advanced cardiac imaging, multidisciplinary evaluation, and diagnostic criteria to diagnose CS. AIMS: To compare the 3 main available diagnostic criteria in patients clinically judged to have CS. METHODS: We prospectively included patients clinically judged to have CS by a multidisciplinary sarcoidosis team from November 2016 to October 2017. We included only incident cases (diagnosis of CS within 1 year of inclusion). We applied retrospectively the following diagnostic criteria: the World Association of Sarcoidosis and Other Granulomatous Diseases (WASOG), the Heart Rhythm Society (HRS), and the Japanese Circulation Society (JCS) 2016 criteria. RESULTS: We identified 69 patients. Diagnostic criteria classified patients as follows: WASOG as highly probable (1.4%), probable (52.2%), possible (0%), some criteria (40.6%), and no criteria (5.8%); HRS as histological diagnosis (1.4%), probable (52.2%), some criteria (40.6%), and no criteria (5.8%); JCS as histological diagnosis (1.4%), clinical diagnosis (58%), some criteria (39.1%), and no criteria (1.4%). Concordance was high between WASOG and HRS (κâ¯=â¯1) but low between JCS and the others (κâ¯=â¯0.326). CONCLUSIONS: A high proportion of patients clinically judged to have CS are unable to be classified according to the 3 main diagnostic criteria. There is low concordance between JCS criteria and the other 2 criteria (WASOG and HRS).
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Cardiomiopatías/diagnóstico , Sarcoidosis/diagnóstico , Adulto , Técnicas de Diagnóstico Cardiovascular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.
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Pulmón , Sistema de Señalización de MAP Quinasas , Activación de Macrófagos , Macrófagos/inmunología , Neumonía Bacteriana , Infecciones por Pseudomonas , Pseudomonas aeruginosa/inmunología , Canales Catiónicos TRPV/inmunología , Animales , Femenino , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Lipopolisacáridos/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/patología , Ratones , Ratones Mutantes , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/inmunología , Neumonía Bacteriana/genética , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/prevención & control , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/prevención & control , Canales Catiónicos TRPV/genéticaRESUMEN
Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor-ß (TGF-ß) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-ß induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-ß-induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-ß stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.
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Membrana Celular/metabolismo , Transdiferenciación Celular , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Miofibroblastos/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Línea Celular , Membrana Celular/genética , Membrana Celular/patología , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Miofibroblastos/patología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Canales Catiónicos TRPV/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a specific type of fibrosing interstitial pneumonia of unknown cause. It is usually chronic and progressive, tends to affect mainly adults over age 60, has a predilection for men, and is often fatal. The condition is still underappreciated by pulmonologists and primary care physicians. This article attempts to close that information gap by reviewing the natural course of IPF and presenting an algorithmic approach to diagnosis and treatment based on evidence-based international guidelines. New treatment options are briefly discussed, to raise awareness of new medications that target pulmonary fibrosis.
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Conocimientos, Actitudes y Práctica en Salud , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/terapia , Médicos de Atención Primaria , Neumólogos , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Ion channels/pumps are essential regulators of organ homeostasis and disease. In the present review, we discuss the role of the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), in cytokine secretion and pulmonary inflammatory diseases such as asthma, cystic fibrosis (CF), and acute lung injury/acute respiratory distress syndrome (ARDS). TRPV4 has been shown to play a role in lung diseases associated with lung parenchymal stretch or stiffness. TRPV4 indirectly mediates hypotonicity-induced smooth muscle contraction and airway remodeling in asthma. Further, the literature suggests that in CF TRPV4 may improve ciliary beat frequency enhancing mucociliary clearance, while at the same time increasing pro-inflammatory cytokine secretion/lung tissue injury. Currently it is understood that the role of TRPV4 in immune cell function and associated lung tissue injury/ARDS may depend on the injury stimulus. Uncovering the downstream mechanisms of TRPV4 action in pulmonary inflammatory diseases is likely important to understanding disease pathogenesis and may lead to novel therapeutics.
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Proteínas Quinasas Activadas por AMP/inmunología , Lesión Pulmonar Aguda/inmunología , Envejecimiento/inmunología , Inmunidad Innata , Choque Hemorrágico/inmunología , Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/patología , Animales , Humanos , Choque Hemorrágico/enzimología , Choque Hemorrágico/patologíaRESUMEN
As long-term smokers undergo computed tomography (CT) to screen for lung cancer, cases of interstitial lung disease are being discovered incidentally. This article explains how to distinguish among the most common forms of interstitial lung disease in this situation and the role of primary care physicians in managing them.
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Detección Precoz del Cáncer , Enfermedades Pulmonares Intersticiales/diagnóstico por imagen , Enfermedades Pulmonares Intersticiales/terapia , Neoplasias Pulmonares/diagnóstico por imagen , Atención Primaria de Salud/métodos , Tomografía Computarizada por Rayos X , Humanos , Hallazgos Incidentales , Fibrosis Pulmonar/diagnóstico por imagen , Derivación y Consulta , FumarRESUMEN
Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.
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Fibroblastos/fisiología , Miosina Tipo II/fisiología , Fibrosis Pulmonar/metabolismo , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Polaridad Celular , Forma de la Célula , Matriz Extracelular/fisiología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Fenotipo , Fibrosis Pulmonar/patologíaRESUMEN
Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.
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Lipopolisacáridos/inmunología , Lesión Pulmonar/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Canales Catiónicos TRPV/inmunología , Animales , Células Cultivadas , Citocinas/biosíntesis , Citocinas/inmunología , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Matriz Extracelular/metabolismo , Inmunoglobulina G/inmunología , Lesión Pulmonar/patología , Fenómenos Mecánicos , Ratones , Ratones Endogámicos C57BL , Microesferas , Fibrosis Pulmonar/inmunología , Transducción de Señal/inmunologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-ß; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-ß1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.
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Diferenciación Celular , Pulmón/metabolismo , Miofibroblastos/metabolismo , Fibrosis Pulmonar/metabolismo , Canales Catiónicos TRPV/biosíntesis , Regulación hacia Arriba , Animales , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/farmacología , Bleomicina/efectos adversos , Bleomicina/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Pulmón/patología , Ratones , Ratones Mutantes , Miofibroblastos/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Canales Catiónicos TRPV/genética , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5ß1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with ß1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5ß1 integrin and uPAR drive the translocation of α5ß1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.