RESUMO
Tissue fibrosis remains unamenable to meaningful therapeutic interventions and is the primary cause of chronic graft failure after organ transplantation. Eukaryotic translation initiation factor (eIF4E), a key translational regulator, serves as convergent target of multiple upstream profibrotic signaling pathways that contribute to mesenchymal cell (MC) activation. Here, we investigate the role of MAP kinase-interacting serine/threonine kinase-induced (MNK-induced) direct phosphorylation of eIF4E at serine 209 (Ser209) in maintaining fibrotic transformation of MCs and determine the contribution of the MNK/eIF4E pathway to the pathogenesis of chronic lung allograft dysfunction (CLAD). MCs from patients with CLAD demonstrated constitutively higher eIF4E phosphorylation at Ser209, and eIF4E phospho-Ser209 was found to be critical in regulating key fibrogenic protein autotaxin, leading to sustained ß-catenin activation and profibrotic functions of CLAD MCs. MNK1 signaling was upregulated in CLAD MCs, and genetic or pharmacologic targeting of MNK1 activity inhibited eIF4E phospho-Ser209 and profibrotic functions of CLAD MCs in vitro. Treatment with an MNK1/2 inhibitor (eFT-508) abrogated allograft fibrosis in an orthotopic murine lung-transplant model. Together these studies identify what we believe is a previously unrecognized MNK/eIF4E/ATX/ß-catenin signaling pathway of fibrotic transformation of MCs and present the first evidence, to our knowledge, for the utility of MNK inhibitors in fibrosis.
Assuntos
Aloenxertos , Fator de Iniciação 4E em Eucariotos , Transplante de Pulmão , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Camundongos , Fosforilação , Humanos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Masculino , Fibrose , Feminino , Transdução de SinaisRESUMO
Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.
Assuntos
Fosfatase 1 de Especificidade Dupla , Pulmão , Proteína Quinase 14 Ativada por Mitógeno , Miofibroblastos , Fibrose Pulmonar , Animais , Camundongos , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Miofibroblastos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/enzimologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/induzido quimicamente , Pulmão/patologia , Pulmão/metabolismo , Bleomicina/toxicidade , Humanos , Camundongos Knockout , Camundongos Transgênicos , ApoptoseRESUMO
Rationale: Chronic lung allograft dysfunction (CLAD) is the leading cause of death after lung transplant, and azithromycin has variable efficacy in CLAD. The lung microbiome is a risk factor for developing CLAD, but the relationship between lung dysbiosis, pulmonary inflammation, and allograft dysfunction remains poorly understood. Whether lung microbiota predict outcomes or modify treatment response after CLAD is unknown. Objectives: To determine whether lung microbiota predict post-CLAD outcomes and clinical response to azithromycin. Methods: Retrospective cohort study using acellular BAL fluid prospectively collected from recipients of lung transplant within 90 days of CLAD onset. Lung microbiota were characterized using 16S rRNA gene sequencing and droplet digital PCR. In two additional cohorts, causal relationships of dysbiosis and inflammation were evaluated by comparing lung microbiota with CLAD-associated cytokines and measuring ex vivo P. aeruginosa growth in sterilized BAL fluid. Measurements and Main Results: Patients with higher bacterial burden had shorter post-CLAD survival, independent of CLAD phenotype, azithromycin treatment, and relevant covariates. Azithromycin treatment improved survival in patients with high bacterial burden but had negligible impact on patients with low or moderate burden. Lung bacterial burden was positively associated with CLAD-associated cytokines, and ex vivo growth of P. aeruginosa was augmented in BAL fluid from transplant recipients with CLAD. Conclusions: In recipients of lung transplants with chronic rejection, increased lung bacterial burden is an independent risk factor for mortality and predicts clinical response to azithromycin. Lung bacterial dysbiosis is associated with alveolar inflammation and may be promoted by underlying lung allograft dysfunction.
Assuntos
Azitromicina , Rejeição de Enxerto , Transplante de Pulmão , Microbiota , Humanos , Azitromicina/uso terapêutico , Masculino , Feminino , Pessoa de Meia-Idade , Rejeição de Enxerto/microbiologia , Rejeição de Enxerto/prevenção & controle , Estudos Retrospectivos , Adulto , Microbiota/efeitos dos fármacos , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Pulmão/microbiologia , Doença Crônica , Transplantados/estatística & dados numéricos , Idoso , Disbiose , Estudos de Coortes , Líquido da Lavagem Broncoalveolar/microbiologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is marked by unremitting matrix deposition and architectural distortion. Multiple profibrotic pathways contribute to the persistent activation of mesenchymal cells (MCs) in fibrosis, highlighting the need to identify and target common signaling pathways. The transcription factor nuclear factor of activated T cells 1 (NFAT1) lies downstream of second messenger calcium signaling and has been recently shown to regulate key profibrotic mediator autotaxin (ATX) in lung MCs. Herein, we investigate the role of NFAT1 in regulating fibroproliferative responses during the development of lung fibrosis. Nfat1-/--deficient mice subjected to bleomycin injury demonstrated improved survival and protection from lung fibrosis and collagen deposition as compared with bleomycin-injured wild-type (WT) mice. Chimera mice, generated by reconstituting bone marrow cells from WT or Nfat1-/- mice into irradiated WT mice (WTâWT and Nfat1-/-âWT), demonstrated no difference in bleomycin-induced fibrosis, suggesting immune influx-independent fibroprotection in Nfat1-/- mice. Examination of lung tissue and flow sorted lineageneg/platelet-derived growth factor receptor alpha (PDGFRα)pos MCs demonstrated decreased MC numbers, proliferation [↓ cyclin D1 and 5-ethynyl-2'-deoxyuridine (EdU) incorporation], myofibroblast differentiation [↓ α-smooth muscle actin (α-SMA)], and survival (↓ Birc5) in Nfat1-/- mice. Nfat1 deficiency abrogated ATX expression in response to bleomycin in vivo and MCs derived from Nfat1-/- mice demonstrated decreased ATX expression and migration in vitro. Human IPF MCs demonstrated constitutive NFAT1 activation, and regulation of ATX in these cells by NFAT1 was confirmed using pharmacological and genetic inhibition. Our findings identify NFAT1 as a critical mediator of profibrotic processes, contributing to dysregulated lung remodeling and suggest its targeting in MCs as a potential therapeutic strategy in IPF.NEW & NOTEWORTHY Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.
Assuntos
Fibrose Pulmonar Idiopática , Pulmão , Animais , Humanos , Camundongos , Bleomicina/farmacologia , Diferenciação Celular/genética , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
BACKGROUND: Small airway inflammation and fibrosis or bronchiolitis obliterans (BO) is the predominant presentation of chronic lung allograft dysfunction (CLAD) post-lung transplantation. Carbon monoxide (CO) is a critical endogenous signaling transducer with known anti-inflammatory and anti-fibrotic effects but its therapeutic potential in CLAD remains to be fully elucidated. METHODS: Here we investigate the effect of inhaled CO in modulating chronic lung allograft rejection pathology in a murine orthotopic lung transplant model of BO (B6D2F1/JâDBA/2J). Additionally, the effects of CO on the activated phenotype of mesenchymal cells isolated from human lung transplant recipients with CLAD were studied. RESULTS: Murine lung allografts treated with CO (250 ppm × 30 minutes twice daily from days 7 to 40 post-transplantation) demonstrated decreased immune cell infiltration, fibrosis, and airway obliteration by flow cytometry, trichrome staining, and morphometric analysis, respectively. Decreased total collagen, with levels comparable to isografts, was noted in CO-treated allografts by quantitative hydroxyproline assay. In vitro, CO (250 ppm × 16h) was effective in reversing the fibrotic phenotype of human CLAD mesenchymal cells with decreased collagen I and ß-catenin expression as well as an inhibitory effect on ERK1/2 MAPK, and mTORC1/2 signaling. Sildenafil, a phosphodiesterase 5 inhibitor, partially mimicked the effects of CO on CLAD mesenchymal cells and was partially effective in decreasing collagen deposition in murine allografts, suggesting the contribution of cGMP-dependent and -independent mechanisms in mediating the effect of CO. CONCLUSION: These results suggest a potential role for CO in alleviating allograft fibrosis and mitigating chronic rejection pathology post-lung transplant.
Assuntos
Bronquiolite Obliterante , Transplante de Pulmão , Humanos , Animais , Camundongos , Monóxido de Carbono , Aloenxertos/patologia , Transplante de Pulmão/efeitos adversos , Fibrose , Pulmão/patologia , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/prevenção & controle , Colágeno , Rejeição de EnxertoRESUMO
In this study, we demonstrate that forkhead box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, was retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as key players in lung allograft remodeling and fibrosis. Using Foxf1-tdTomato BAC (Foxf1-tdTomato) and Foxf1-tdTomato Col1a1-GFP mice, we show that Lin-Foxf1+ cells encompassed the stem cell antigen 1+CD34+ (Sca1+CD34+) subset of collagen 1-expressing mesenchymal cells (MCs) with a capacity to generate CFU and lung epithelial organoids. Histologically, FOXF1-expressing MCs formed a 3D network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA-Seq confirmed distinct transcriptional signatures of Foxf1+ and Foxf1- MCs, with Foxf1-expressing cells delineated by their high expression of the transcription factor glioma-associated oncogene 1 (Gli1) and low expression of integrin α8 (Itga), versus other collagen-expressing MCs. FOXF1+Gli1+ MCs showed proximity to Sonic hedgehog-expressing (Shh-expressing) bronchial epithelium, and mesenchymal expression of Foxf1 and Gli1 was found to be dependent on paracrine Shh signaling in epithelial organoids. Using a murine lung transplant model, we show dysregulation of epithelial-mesenchymal SHH/GLI1/FOXF1 crosstalk and expansion of this specific peribronchial MC population in chronically rejecting fibrotic lung allografts.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto/metabolismo , Transplante de Pulmão , Células-Tronco Mesenquimais/metabolismo , Alvéolos Pulmonares/metabolismo , Fibrose Pulmonar/metabolismo , Aloenxertos , Animais , Doença Crônica , Fatores de Transcrição Forkhead/genética , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologiaRESUMO
BACKGROUND: Alterations in the respiratory microbiome are common in chronic lung diseases, correlate with decreased lung function, and have been associated with disease progression. The clinical significance of changes in the respiratory microbiome after lung transplant, specifically those related to development of chronic lung allograft dysfunction (CLAD), are unknown. The aim of this study was to evaluate the effect of lung microbiome characteristics in healthy lung transplant recipients on subsequent CLAD-free survival. METHODS: We prospectively studied a cohort of lung transplant recipients at the University of Michigan (Ann Arbor, MI, USA). We analysed characteristics of the respiratory microbiome in acellular bronchoalveolar lavage fluid (BALF) collected from asymptomatic patients during per-protocol surveillance bronchoscopy 1 year after lung transplantation. For our primary endpoint, we evaluated a composite of development of CLAD or death at 500 days after the 1-year surveillance bronchoscopy. Our primary microbiome predictor variables were bacterial DNA burden (total 16S rRNA gene copies per mL of BALF, quantified via droplet digital PCR) and bacterial community composition (determined by bacterial 16S rRNA gene sequencing). Patients' lung function was followed serially at least every 3 months by spirometry, and CLAD was diagnosed according to International Society of Heart and Lung Transplant 2019 guidelines. FINDINGS: We analysed BALF from 134 patients, collected during 1-year post-transplant surveillance bronchoscopy between Oct 21, 2005, and Aug 25, 2017. Within 500 days of follow-up from the time of BALF sampling, 24 (18%) patients developed CLAD, five (4%) died before confirmed development of CLAD, and 105 (78%) patients remained CLAD-free with complete follow-up. Lung bacterial burden was predictive of CLAD development or death within 500 days of the surveillance bronchoscopy, after controlling for demographic and clinical factors, including immunosuppression and bacterial culture results, in a multivariable survival model. This relationship was evident when burden was analysed as a continuous variable (per log10 increase in burden, HR 2·49 [95% CI 1·38-4·48], p=0·0024) or by tertiles (middle vs lowest bacterial burden tertile, HR 4·94 [1·25-19·42], p=0·022; and highest vs lowest, HR 10·56 [2·53-44·08], p=0·0012). In patients who developed CLAD or died, composition of the lung bacterial community significantly differed to that in patients who survived and remained CLAD-free (on permutational multivariate analysis of variance, p=0·047 at the taxonomic level of family), although differences in community composition were associated with bacterial burden. No individual bacterial taxa were definitively associated with CLAD development or death. INTERPRETATION: Among asymptomatic lung transplant recipients at 1-year post-transplant, increased lung bacterial burden is predictive of chronic rejection and death. The lung microbiome represents an understudied and potentially modifiable risk factor for lung allograft dysfunction. FUNDING: US National Institutes of Health, Cystic Fibrosis Foundation, Brian and Mary Campbell and Elizabeth Campbell Carr research gift fund.
Assuntos
Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/microbiologia , Transplante de Pulmão , Pulmão/microbiologia , Microbiota , Transplantados/estatística & dados numéricos , Doença Crônica , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Histopathologic examination of lungs afflicted by chronic lung allograft dysfunction (CLAD) consistently shows both mononuclear cell (MNC) inflammation and mesenchymal cell (MC) fibroproliferation. We hypothesize that interleukin 6 (IL-6) trans-signaling may be a critical mediator of MNC-MC crosstalk and necessary for the pathogenesis of CLAD. Bronchoalveolar lavage (BAL) fluid obtained after the diagnosis of CLAD has approximately twofold higher IL-6 and soluble IL-6 receptor (sIL-6R) levels compared to matched pre-CLAD samples. Human BAL-derived MCs do not respond to treatment with IL-6 alone but have rapid and prolonged JAK2-mediated STAT3 Tyr705 phosphorylation when exposed to the combination of IL-6 and sIL-6R. STAT3 phosphorylation within MCs upregulates numerous genes causing increased invasion and fibrotic differentiation. MNC, a key source of both IL-6 and sIL-6R, produce minimal amounts of these proteins at baseline but significantly upregulate production when cocultured with MCs. Finally, the use of an IL-6 deficient recipient in a murine orthotopic transplant model of CLAD reduces allograft fibrosis by over 50%. Taken together these results support a mechanism where infiltrating MNCs are stimulated by resident MCs to release large quantities of IL-6 and sIL-6R which then feedback onto the MCs to increase invasion and fibrotic differentiation.
Assuntos
Interleucina-6 , Transplante de Pulmão , Aloenxertos , Animais , Fibrose , Humanos , Pulmão/patologia , Transplante de Pulmão/efeitos adversos , Camundongos , Receptores de Interleucina-6RESUMO
Forkhead box F1 (FOXF1) is a lung embryonic mesenchyme-associated transcription factor that demonstrates persistent expression into adulthood in mesenchymal stromal cells. However, its biologic function in human adult lung-resident mesenchymal stromal cells (LR-MSCs) remain to be elucidated. Here, we demonstrate that FOXF1 expression acts as a restraint on the migratory function of LR-MSCs via its role as a novel transcriptional repressor of autocrine motility-stimulating factor Autotaxin (ATX). Fibrotic human LR-MSCs demonstrated lower expression of FOXF1 mRNA and protein, compared to non-fibrotic controls. RNAi-mediated FOXF1 silencing in LR-MSCs was associated with upregulation of key genes regulating proliferation, migration, and inflammatory responses and significantly higher migration were confirmed in FOXF1-silenced LR-MSCs by Boyden chamber. ATX is a secreted lysophospholipase D largely responsible for extracellular lysophosphatidic acid (LPA) production, and was among the top ten upregulated genes upon Affymetrix analysis. FOXF1-silenced LR-MSCs demonstrated increased ATX activity, while mFoxf1 overexpression diminished ATX expression and activity. The FOXF1 silencing-induced increase in LR-MSC migration was abrogated by genetic and pharmacologic targeting of ATX and LPA1 receptor. Chromatin immunoprecipitation analyses identified three putative FOXF1 binding sites in the 1.5 kb ATX promoter which demonstrated transcriptional repression of ATX expression. Together these findings identify FOXF1 as a novel transcriptional repressor of ATX and demonstrate that loss of FOXF1 promotes LR-MSC migration via the ATX/LPA/LPA1 signaling axis.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Sítios de Ligação/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Ontologia Genética , Inativação Gênica , Humanos , Pulmão/citologia , Camundongos , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais/genética , Ativação Transcricional/genética , Regulação para CimaRESUMO
Understanding the distinct pathogenic mechanisms that culminate in allograft fibrosis and chronic graft failure is key in improving outcomes after solid organ transplantation. Here, we describe an F1 â parent orthotopic lung transplant model of restrictive allograft syndrome (RAS), a particularly fulminant form of chronic lung allograft dysfunction (CLAD), and identify a requisite pathogenic role for humoral immune responses in development of RAS. B6D2F1/J (H2-b/d) donor lungs transplanted into the parent C57BL/6J (H2-b) recipients demonstrated a spectrum of histopathologic changes, ranging from lymphocytic infiltration, fibrinous exudates, and endothelialitis to peribronchial and pleuroparenchymal fibrosis, similar to those noted in the human RAS lungs. Gene expression profiling revealed differential humoral immune cell activation as a key feature of the RAS murine model, with significant B cell and plasma cell infiltration noted in the RAS lung allografts. B6D2F1/J lung allografts transplanted into µMt-/- (mature B cell deficient) or activation-induced cytidine deaminase (AID)/secretory µ-chain (µs) double-KO (AID-/-µs-/-) C57BL/6J mice demonstrated significantly decreased allograft fibrosis, indicating a key role for antibody secretion by B cells in mediating RAS pathology. Our study suggests that skewing of immune responses determines the diverse allograft remodeling patterns and highlights the need to develop targeted therapies for specific CLAD phenotypes.
Assuntos
Aloenxertos/imunologia , Aloenxertos/patologia , Imunidade Humoral/imunologia , Animais , Fibrose , Rejeição de Enxerto/imunologia , Pulmão/patologia , Transplante de Pulmão/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Órgãos , FenótipoRESUMO
BACKGROUND: Chronic lung allograft dysfunction (CLAD), the primary cause of poor outcome after lung transplantation, arises from fibrotic remodeling of the allograft and presents as diverse clinical phenotypes with variable courses. Here, we investigate whether bronchoalveolar lavage (BAL) mesenchymal cell activity at CLAD onset can inform regarding disease phenotype, progression, and survival. METHODS: Mesenchymal cell colony-forming units (CFUs) were measured in BAL obtained at CLAD onset (nâ¯=â¯77) and CLAD-free time post-transplant matched controls (nâ¯=â¯77). CFU counts were compared using Wilcoxon's rank-sum test. Cox proportional hazards and restricted means models were utilized to investigate post-CLAD survival. RESULTS: Higher mesenchymal CFU counts were noted in BAL at the time of CLAD onset than in CLAD-free controls. Patients with restrictive allograft syndrome had higher BAL mesenchymal CFU count at CLAD onset than patients with bronchiolitis obliterans syndrome (pâ¯=â¯0.011). Patients with high mesenchymal CFU counts (≥10) at CLAD onset had worse outcomes than those with low (<10) CFU counts, with shorter average survival (2.64 years vs 4.25 years; pâ¯=â¯0.027) and shorter progression-free survival, defined as time to developing either CLAD Stage 3 or death (0.97 years vs 2.70 years; p < 0.001). High CFU count remained predictive of decreased overall survival and progression-free survival after accounting for the CLAD phenotype and other clinical factors in multivariable analysis. CONCLUSIONS: Fulminant fibroproliferation with higher mesenchymal CFU counts in BAL is noted in restrictive allograft syndrome and is independently associated with poor survival after CLAD onset.
Assuntos
Bronquiolite Obliterante/cirurgia , Líquido da Lavagem Broncoalveolar/citologia , Transplante de Pulmão , Células-Tronco Mesenquimais/citologia , Disfunção Primária do Enxerto/etiologia , Adulto , Aloenxertos , Broncoscopia , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Disfunção Primária do Enxerto/diagnóstico , Disfunção Primária do Enxerto/mortalidade , Estudos Prospectivos , Taxa de Sobrevida/tendências , Estados Unidos/epidemiologiaRESUMO
Mammalian target of rapamycin complex 2 (mTORC2) has been shown to regulate mTORC1/4E-BP1/eIF4E signaling and collagen I expression in mesenchymal cells (MCs) during fibrotic activation. Here we investigated the regulation of the mTORC2 binding partner mammalian stress-activated protein kinase-interacting protein 1 (mSin1) in MCs derived from human lung allografts and identified a novel role for mSin1 during fibrosis. mSin1 was identified as a common downstream target of key fibrotic pathways, and its expression was increased in MCs in response to pro-fibrotic mediators: lysophosphatidic acid (LPA), transforming growth factor ß, and interleukin 13. Fibrotic MCs had higher mSin1 protein levels than nonfibrotic MCs, and siRNA-mediated silencing of mSIN1 inhibited collagen I expression and mTORC1/2 activity in these cells. Autocrine LPA signaling contributed to constitutive up-regulation of mSin1 in fibrotic MCs, and mSin1 was decreased because of LPA receptor 1 siRNA treatment. We identified c-Jun N-terminal kinase (JNK) as a key intermediary in mSin1 up-regulation by the pro-fibrotic mediators, as pharmacological and siRNA-mediated inhibition of JNK prevented the LPA-induced mSin1 increase. Proteasomal inhibition rescued mSin1 levels after JNK inhibition in LPA-treated MCs, and the decrease in mSin1 ubiquitination in response to LPA was counteracted by JNK inhibitors. Constitutive JNK1 overexpression induced mSin1 expression and could drive mTORC2 and mTORC1 activation and collagen I expression in nonfibrotic MCs, effects that were reversed by siRNA-mediated mSIN1 silencing. These results indicate that LPA stabilizes mSin1 protein expression via JNK signaling by blocking its proteasomal degradation and identify the LPA/JNK/mSin1/mTORC/collagen I pathway as critical for fibrotic activation of MCs.
Assuntos
Proteínas de Transporte/metabolismo , Fibrose/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mesoderma/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibrose/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Pulmão/citologia , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Mesoderma/citologia , Proteínas Monoméricas de Ligação ao GTP , Fosforilação , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de SinaisRESUMO
Tissue fibrosis is the primary cause of long-term graft failure after organ transplantation. In lung allografts, progressive terminal airway fibrosis leads to an irreversible decline in lung function termed bronchiolitis obliterans syndrome (BOS). Here, we have identified an autocrine pathway linking nuclear factor of activated T cells 2 (NFAT1), autotaxin (ATX), lysophosphatidic acid (LPA), and ß-catenin that contributes to progression of fibrosis in lung allografts. Mesenchymal cells (MCs) derived from fibrotic lung allografts (BOS MCs) demonstrated constitutive nuclear ß-catenin expression that was dependent on autocrine ATX secretion and LPA signaling. We found that NFAT1 upstream of ATX regulated expression of ATX as well as ß-catenin. Silencing NFAT1 in BOS MCs suppressed ATX expression, and sustained overexpression of NFAT1 increased ATX expression and activity in non-fibrotic MCs. LPA signaling induced NFAT1 nuclear translocation, suggesting that autocrine LPA synthesis promotes NFAT1 transcriptional activation and ATX secretion in a positive feedback loop. In an in vivo mouse orthotopic lung transplant model of BOS, antagonism of the LPA receptor (LPA1) or ATX inhibition decreased allograft fibrosis and was associated with lower active ß-catenin and dephosphorylated NFAT1 expression. Lung allografts from ß-catenin reporter mice demonstrated reduced ß-catenin transcriptional activation in the presence of LPA1 antagonist, confirming an in vivo role for LPA signaling in ß-catenin activation.
Assuntos
Comunicação Autócrina , Lisofosfolipídeos/fisiologia , Fibrose Pulmonar/metabolismo , beta Catenina/metabolismo , Aloenxertos , Animais , Bronquiolite Obliterante/metabolismo , Células Cultivadas , Colágeno/biossíntese , Feminino , Rejeição de Enxerto/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Transplante de Pulmão , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fatores de Transcrição NFATC/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Ativação TranscricionalRESUMO
Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states.
Assuntos
Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Pulmão/patologia , Transplante de Pulmão , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Morfolinas/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
AIM: The aim of this study was to determine the relationship between parent and child Full-scale IQ (FSIQ) in children with epilepsy and in typically developing comparison children and to examine parent-child IQ differences by epilepsy characteristics. METHOD: The study participants were 97 children (50 males, 47 females; age range 8-18y; mean age 12y 3mo, SD 3y1mo) with recent-onset epilepsy including idiopathic generalized (n=43) and idiopathic localization-related epilepsies (n=54); 69 healthy comparison children (38 females, 31 males; age range 8-18y; mean age 12y 8mo, SD 3y 2mo), and one biological parent per child. All participants were administered the Wechsler Abbreviated Scale of Intelligence (WASI). FSIQ was compared in children with epilepsy and typically developing children; FSIQ was compared in the parents of typically developing children and the parents of participants with epilepsy; parent-child FSIQ differences were compared between the groups. RESULTS: FSIQ was lower in children with epilepsy than in comparison children (p<0.001). FSIQ of parents of children with epilepsy did not differ from the FSIQ of the parents of typically developing children. Children with epilepsy had significantly lower FSIQ than their parents (p<0.001), whereas comparison children did not. The parent-child IQ difference was significantly higher in the group with epilepsy than the comparison group (p=0.043). Epilepsy characteristics were not related to parent-child IQ difference. INTERPRETATION: Parent-child IQ difference appears to be a marker of epilepsy impact independent of familial IQ, epilepsy syndrome, and clinical seizure features. This marker is evident early in the course of idiopathic epilepsies and can be tracked over time.
Assuntos
Epilepsia/fisiopatologia , Inteligência/fisiologia , Adolescente , Adulto , Biomarcadores , Estudos de Casos e Controles , Criança , Epilepsias Parciais/fisiopatologia , Feminino , Humanos , Masculino , Pais/psicologia , Escalas de WechslerRESUMO
OBJECTIVE: To characterize neuropsychological and academic status in children, ages 8-18 years, with new-/recent-onset idiopathic generalized epilepsy (IGE) and idiopathic localization-related epilepsy (ILRE) compared with healthy controls. STUDY DESIGN: Participants underwent neuropsychological assessment, and parents were interviewed regarding their child's academic history. Cognitive scores for children with epilepsy were age- and sex-adjusted and compared with controls across both broad-band (IGE n = 41 and ILRE n = 53) and narrow-band (childhood/juvenile absence, juvenile myoclonic, benign epilepsy with centro-temporal spikes, and focal [temporal/frontal/not otherwise specified]) syndromes. Academic histories were examined, including problems antecedent to epilepsy onset and diagnosis. RESULTS: Children with new/recent-onset epilepsies exhibit considerable cognitive abnormality at baseline, including patterns of shared abnormalities across syndromes (eg, psychomotor slowing) as well as unique syndrome-specific cognitive effects (eg, executive function in IGE and language/verbal memory in ILRE) that are observed and sometimes exacerbated in specific IGE and ILRE syndromes. Academic difficulties are evident in approximately 50% of the children with epilepsy, affecting all syndrome groups to an equal degree. DISCUSSION: Patterns of shared and syndrome-specific cognitive abnormalities and academic problems are present early in the course of virtually all epilepsy syndromes examined here, including syndromes classically viewed as benign. This is the base upon which the effects of recurrent seizures, treatment, and psychosocial effects will be added over time.
Assuntos
Transtornos Cognitivos/epidemiologia , Epilepsia Generalizada/psicologia , Deficiências da Aprendizagem/epidemiologia , Adolescente , Criança , Transtornos Cognitivos/complicações , Epilepsia Generalizada/complicações , Feminino , Humanos , Deficiências da Aprendizagem/complicações , Masculino , Testes NeuropsicológicosRESUMO
RATIONALE: Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood. OBJECTIVES: This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs. METHODS: Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists. MEASUREMENT AND MAIN RESULTS: PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1ß was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1ß. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio. CONCLUSIONS: These data identify the PGE2 axis as an important autocrine-paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.
Assuntos
Bronquiolite Obliterante/metabolismo , Bronquiolite Obliterante/patologia , Dinoprostona/metabolismo , Transplante de Pulmão , Pulmão/metabolismo , Pulmão/patologia , Actinas/metabolismo , Adenilil Ciclases/metabolismo , Western Blotting , Líquido da Lavagem Broncoalveolar , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fibrose , Humanos , Interleucina-1beta/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Multipotent mesenchymal progenitor cells, termed "mesenchymal stem cells" (MSCs), have been demonstrated to reside in human adult lungs. However, there is little information regarding the associations of these local mesenchymal progenitors with other resident somatic cells and their potential for therapeutic use. Here we provide in vivo and in vitro evidence for the ability of human adult lung-resident MSCs (LR-MSCs) to interact with the local epithelial cells. The in vivo retention and localization of human LR-MSCs in an alveolar microenvironment was investigated by placing PKH-26 or DsRed lentivirus-labeled human LR-MSCs in the lungs of immunodeficient (SCID) mice. At 3 weeks after intratracheal administration, 19.3 ± 3.21% of LR-MSCs were recovered, compared with 3.47 ± 0.51% of control fibroblasts, as determined by flow cytometry. LR-MSCs were found to persist in murine lungs for up to 6 months and demonstrated preferential localization to the corners of the alveoli in close proximity to type II alveolar epithelial cells, the progenitor cells of the alveolar epithelium. In vitro, LR-MSCs established gap junction communications with lung alveolar and bronchial epithelial cells and demonstrated an ability to secrete keratinocyte growth factor, an important modulator of epithelial cell proliferation and differentiation. Gap junction communications were also demonstrable between LR-MSCs and resident murine cells in vivo. This study demonstrates, for the first time, an ability of tissue-specific MSCs to engraft in their organ of origin and establishes a pathway of bidirectional interaction between these mesenchymal progenitors and adult somatic epithelial cells in the lung.
Assuntos
Células Epiteliais Alveolares/metabolismo , Comunicação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Alvéolos Pulmonares/cirurgia , Animais , Separação Celular/métodos , Rastreamento de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Junções Comunicantes/metabolismo , Vetores Genéticos , Sobrevivência de Enxerto , Humanos , Lentivirus/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Camundongos SCID , Compostos Orgânicos/metabolismo , Alvéolos Pulmonares/metabolismo , Fatores de Tempo , Transfecção , Proteína Vermelha FluorescenteRESUMO
RATIONALE: Bronchoalveolar lavage fluid (BAL) from human lung allografts demonstrates the presence of a multipotent mesenchymal stromal cell population. However, the clinical relevance of this novel cellular component of BAL and its association with bronchiolitis obliterans syndrome (BOS), a disease marked by progressive airflow limitation secondary to fibrotic obliteration of the small airways, remains to be determined. OBJECTIVES: In this study we investigate the association of number of mesenchymal stromal cells in BAL with development of BOS in human lung transplant recipients. METHODS: Mesenchymal colony-forming units (CFUs) were quantitated in a cohort of 405 BAL samples obtained from 162 lung transplant recipients. Poisson generalized estimating equations were used to determine the predictors of BAL mesenchymal CFU count. MEASUREMENTS AND MAIN RESULTS: Higher CFU counts were noted early post-transplantation; time from transplant to BAL of greater than 3 months predicted 0.4-fold lower CFU counts (P = 0.0001). BOS diagnosis less than or equal to 365 days before BAL was associated with a 2.11-fold higher CFU count (P = 0.02). There were 2.62- and 2.70-fold higher CFU counts noted in the presence of histologic diagnosis of bronchiolitis obliterans (P = 0.05) and organizing pneumonia (0.0003), respectively. In BAL samples obtained from BOS-free patients greater than 6 months post-transplantation (n = 173), higher mesenchymal CFU counts (≥10) significantly predicted BOS onset in both univariate (hazard ratio, 5.61; 95% CI, 3.03-10.38; P < 0.0001) and multivariate (hazard ratio, 5.02; 95% CI, 2.40-10.51; P < 0.0001) Cox regression analysis. CONCLUSIONS: Measurement of mesenchymal CFUs in the BAL provides predictive information regarding future BOS onset.