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BACKGROUND: Integrative multiomics can elucidate pulmonary arterial hypertension (PAH) pathobiology, but procuring human PAH lung samples is rare. METHODS: We leveraged transcriptomic profiling and deep phenotyping of the largest multicenter PAH lung biobank to date (96 disease and 52 control) by integration with clinicopathologic data, genome-wide association studies, Bayesian regulatory networks, single-cell transcriptomics, and pharmacotranscriptomics. RESULTS: We identified 2 potentially protective gene network modules associated with vascular cells, and we validated ASPN, coding for asporin, as a key hub gene that is upregulated as a compensatory response to counteract PAH. We found that asporin is upregulated in lungs and plasma of multiple independent PAH cohorts and correlates with reduced PAH severity. We show that asporin inhibits proliferation and transforming growth factor-ß/phosphorylated SMAD2/3 signaling in pulmonary artery smooth muscle cells from PAH lungs. We demonstrate in Sugen-hypoxia rats that ASPN knockdown exacerbated PAH and recombinant asporin attenuated PAH. CONCLUSIONS: Our integrative systems biology approach to dissect the PAH lung transcriptome uncovered asporin as a novel protective target with therapeutic potential in PAH.
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Humans living at high-altitude (HA) have adapted to this environment by increasing pulmonary vascular and alveolar growth. RNA sequencing data from a novel murine model that mimics this phenotypical response to HA suggested estrogen signaling via estrogen receptor alpha (ERα) may be involved in this adaptation. We hypothesized ERα was a key mediator in the cardiopulmonary adaptation to chronic hypoxia and sought to delineate the mechanistic role ERα contributes to this process by exposing novel loss-of-function ERα mutant (ERαMut) rats to simulated HA. ERα mutant or wild-type (wt) rats were exposed to normoxia or hypoxia starting at conception and continued postnatally until 6 wk of age. Both wt and ERαMut animals born and raised in hypoxia exhibited lower body mass and higher hematocrits, total alveolar volumes (Va), diffusion capacities of carbon monoxide (DLCO), pulmonary arteriole (PA) wall thickness, and Fulton indices than normoxia animals. Right ventricle adaptation was maintained in the setting of hypoxia. Although no major physiologic differences were seen between wt and ERαMut animals at either exposure, ERαMut animals exhibited smaller mean linear intercepts (MLI) and increased PA total and lumen areas. Hypoxia exposure or ERα loss-of-function did not affect lung mRNA abundance of vascular endothelial growth factor, angiopoietin 2, or apelin. Sexual dimorphisms were noted in PA wall thickness and PA lumen area in ERαMut rats. In summary, in room air-exposed rats and rats with peri- and postnatal hypoxia exposure, ERα loss-of-function was associated with decreased alveolar size (primarily driven by hypoxic animals) and increased PA remodeling.NEW & NOTEWORTHY By exposing novel loss-of-function estrogen receptor alpha (Erα) mutant rats to a novel model of human high-altitude exposure, we demonstrate that ERα has subtle but inconsistent effects on endpoints relevant to cardiopulmonary adaptation to chronic hypoxia. Given that we observed some histologic, sex, and genotype differences, further research into cell-specific effects of ERα during hypoxia-induced cardiopulmonary adaptation is warranted.
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Adaptación Fisiológica , Receptor alfa de Estrógeno , Hipoxia , Animales , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Femenino , Hipoxia/metabolismo , Hipoxia/fisiopatología , Ratas , Masculino , Pulmón/metabolismo , Pulmón/patología , Altitud , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.
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CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.
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Fibrosis Pulmonar Idiopática , Lesión Pulmonar , Animales , Bleomicina , Antígenos CD55/genética , Antígenos CD55/metabolismo , Cadherinas , Caspasa 3/metabolismo , Complemento C3a , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento , Fibrosis , Glicosilfosfatidilinositoles , Proteínas de Choque Térmico , Humanos , Fibrosis Pulmonar Idiopática/patología , Lesión Pulmonar/inducido químicamente , Ratones , Toxina del Pertussis , ARN Mensajero , ARN Interferente Pequeño , TunicamicinaRESUMEN
Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer.
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Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/genética , Lesiones Precancerosas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma del Pulmón/inducido químicamente , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Femenino , Captura por Microdisección con Láser/métodos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Reacción en Cadena de la Polimerasa/métodos , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Secuenciación Completa del Genoma/métodosRESUMEN
Mechanisms driving adaptive developmental responses to chronic high-altitude (HA) exposure are incompletely known. We developed a novel rat model mimicking the human condition of cardiopulmonary adaptation to HA starting at conception and spanning the in utero and postnatal timeframe. We assessed lung growth and cardiopulmonary structure and function and performed transcriptome analyses to identify mechanisms facilitating developmental adaptations to chronic hypoxia. To generate the model, breeding pairs of Sprague-Dawley rats were exposed to hypobaric hypoxia (equivalent to 9,000 ft elevation). Mating, pregnancy, and delivery occurred in hypoxic conditions. Six weeks postpartum, structural and functional data were collected in the offspring. RNA-Seq was performed on right ventricle (RV) and lung tissue. Age-matched breeding pairs and offspring under room air (RA) conditions served as controls. Hypoxic rats exhibited significantly lower body weights and higher hematocrit levels, alveolar volumes, pulmonary diffusion capacities, RV mass, and RV systolic pressure, as well as increased pulmonary artery remodeling. RNA-Seq analyses revealed multiple differentially expressed genes in lungs and RVs from hypoxic rats. Although there was considerable similarity between hypoxic lungs and RVs compared with RA controls, several upstream regulators unique to lung or RV were identified. We noted a pattern of immune downregulation and regulation patterns of immune and hormonal mediators similar to the genome from patients with pulmonary arterial hypertension. In summary, we developed a novel murine model of chronic hypoxia exposure that demonstrates functional and structural phenotypes similar to human adaptation. We identified transcriptomic alterations that suggest potential mechanisms for adaptation to chronic HA.
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Adaptación Fisiológica/fisiología , Altitud , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Transcriptoma/fisiología , Animales , Modelos Animales de Enfermedad , Pulmón/fisiopatología , Ratas Sprague-Dawley , Remodelación Vascular/fisiologíaRESUMEN
BACKGROUND: Primary graft dysfunction (PGD) is the leading cause of early mortality after lung transplantation. Anti-collagen type-V (col(V)) immunity has been observed in animal models of ischemia-reperfusion injury (IRI) and in PGD. We hypothesized that collagen type-V is an innate danger signal contributing to PGD pathogenesis. METHODS: Anti-col(V) antibody production was detected by flow cytometric assay following cultures of murine CD19+ splenic cells with col.(V). Responding murine B cells were phenotyped using surface markers. RNA-Seq analysis was performed on murine CD19+ cells. Levels of anti-col(V) antibodies were measured in 188 recipients from the Lung Transplant Outcomes Group (LTOG) after transplantation. RESULTS: Col(V) induced rapid production of anti-col(V) antibodies from murine CD19+ B cells. Subtype analysis demonstrated innate B-1 B cells bound col.(V). Col(V) induced a specific transcriptional signature in CD19+ B cells with similarities to, yet distinct from, B cell receptor (BCR) stimulation. Rapid de novo production of anti-col(V) Abs was associated with an increased incidence of clinical PGD after lung transplant. CONCLUSIONS: This study demonstrated that col.(V) is an rapidly recognized by B cells and has specific transcriptional signature. In lung transplants recipients the rapid seroconversion to anti-col(V) Ab is linked to increased risk of grade 3 PGD.
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Subgrupos de Linfocitos B/fisiología , Colágeno Tipo V/inmunología , Rechazo de Injerto/inmunología , Trasplante de Pulmón , Adulto , Anciano , Animales , Formación de Anticuerpos , Antígenos CD19/metabolismo , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Inmunidad Innata , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , TranscriptomaAsunto(s)
Factores de Intercambio de Guanina Nucleótido/genética , Síndrome de Hermanski-Pudlak/genética , Proteínas de la Membrana/genética , Fibrosis Pulmonar/genética , Mutación del Sistema de Lectura , Factores de Intercambio de Guanina Nucleótido/metabolismo , Haplotipos , Síndrome de Hermanski-Pudlak/complicaciones , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mutación Missense , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismoRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary artery pressure and vascular resistance, typically leading to right heart failure and death. Current therapies improve quality of life of the patients but have a modest effect on long-term survival. A detailed transcriptomics and systems biology view of the PAH lung is expected to provide new testable hypotheses for exploring novel treatments. We completed transcriptomics analysis of PAH and control lung tissue to develop disease-specific and clinical data/tissue pathology gene expression classifiers from expression datasets. Gene expression data were integrated into pathway analyses. Gene expression microarray data were collected from 58 PAH and 25 control lung tissues. The strength of the dataset and its derived disease classifier was validated using multiple approaches. Pathways and upstream regulators analyses was completed with standard and novel graphical approaches. The PAH lung dataset identified expression patterns specific to PAH subtypes, clinical parameters, and lung pathology variables. Pathway analyses indicate the important global role of TNF and transforming growth factor signaling pathways. In addition, novel upstream regulators and insight into the cellular and innate immune responses driving PAH were identified. Finally, WNT-signaling pathways may be a major determinant underlying the observed sex differences in PAH. This study provides a transcriptional framework for the PAH-diseased lung, supported by previously reported findings, and will be a valuable resource to the PAH research community. Our investigation revealed novel potential targets and pathways amenable to further study in a variety of experimental systems.
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Pulmón/metabolismo , Pulmón/patología , Hipertensión Arterial Pulmonar/genética , Análisis de Sistemas , Transcriptoma/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Lactante , Masculino , Persona de Mediana Edad , Hipertensión Arterial Pulmonar/patología , Caracteres Sexuales , Transducción de Señal/genética , Adulto JovenRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0076451.].
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Interleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-ß in murine bronchiolitis obliterans; that TGF-ß and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.
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Bleomicina/farmacología , Activación de Complemento/efectos de los fármacos , Fibrosis/metabolismo , Interleucina-17/deficiencia , Interleucina-17/metabolismo , Enfermedades Pulmonares/metabolismo , Anciano , Animales , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosis/genética , Técnica del Anticuerpo Fluorescente , Hemólisis/genética , Hemólisis/fisiología , Humanos , Interleucina-17/genética , Enfermedades Pulmonares/genética , Masculino , Ratones , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-ß signaling, the specific hierarchical interactions between TGF-ß and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-ß-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-ß (Ad-TGFß) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-ß-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFß-injured lungs: we detected the recovery of Smad7 (TGF-ß inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-ß-mediated loss of DAF was prevented. Conversely, blockade of the TGF-ß receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-ß caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-ß activates complement and promotes lung fibrosis.
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Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.
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Antígenos CD55/metabolismo , Epitelio/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/metabolismo , Aminoácidos Dicarboxílicos/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Masculino , Ratones Endogámicos C57BLRESUMEN
Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-ß and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-ß1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-ß/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.
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Fibroblastos/metabolismo , Fibrosis Pulmonar/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Línea Celular , Cadena alfa 1 del Colágeno Tipo I , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica/fisiología , Humanos , Lesión Pulmonar/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fibrosis Pulmonar/inducido químicamente , Interferencia de ARN , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/genética , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor ß1 (TGF-ß1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-ß1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-ß1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-ß1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-ß), and whereas TGF-ß1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-ß1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-ß1 expression
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Activación de Complemento , Fibrosis Pulmonar Idiopática/metabolismo , Mucosa Respiratoria/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Anciano , Antígenos CD55/genética , Antígenos CD55/metabolismo , Células Cultivadas , Femenino , Humanos , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/patología , Masculino , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Persona de Mediana Edad , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Proteína smad7/genética , Proteína smad7/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. METHODS: Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. RESULTS: Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-ß, Il-1ß, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-ß superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). CONCLUSIONS: Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- ß-related signaling pathways.
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Colágeno Tipo V/inmunología , Tolerancia Inmunológica , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Bleomicina/efectos adversos , Colágeno Tipo I/inmunología , Colágeno Tipo V/administración & dosificación , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Ratones , Nebulizadores y Vaporizadores , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.
Asunto(s)
Bronquiolitis Obliterante/inmunología , Activación de Complemento , Rechazo de Injerto/inmunología , Interleucina-17/metabolismo , Trasplante de Pulmón/efectos adversos , Animales , Autoinmunidad , Líquido del Lavado Bronquioalveolar , Antígenos CD55/biosíntesis , Colágeno Tipo V/inmunología , Complemento C3a/biosíntesis , Complemento C5 , Regulación hacia Abajo , Humanos , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucina-6/biosíntesis , Prueba de Cultivo Mixto de Linfocitos , Proteína Cofactora de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptores de Complemento/biosíntesis , Receptores de Complemento 3bRESUMEN
Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2, or MK2), a serine/threonine kinase downstream of p38 mitogen-activated protein kinase, has been implicated in inflammation and fibrosis. Compared with pathologically normal lung tissue, significantly higher concentrations of activated MK2 are evident in lung biopsies of patients with idiopathic pulmonary fibrosis (IPF). Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust, activated MK2 expression on Day 7 (prefibrotic stage) and Day 14 (postfibrotic stage). To determine the effects of MK2 inhibition during the postinflammatory/prefibrotic and postfibrotic stages, C57BL/6 mice received intratracheal bleomycin instillation (0.025 U; Day 0), followed by PBS or the MK2 inhibitor (MK2i; 37.5 µg/kg), administered via either local (nebulized) or systemic (intraperitoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning on either Day 7 or Day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 concentrations, and modulated the local mRNA expression of profibrotic cytokine il-1ß, matrix-related genes col1a2, col3a1, and lox, and transforming growth factor-ß family members, including smad3, serpine1 (pai1), and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, the secretion of collagen Type I, fibronectin, and the activation of focal adhesion kinase, whereas activated MK2 was attenuated at optimal doses. The peptide-mediated inhibition of MK2 affects both inflammatory and fibrotic responses, and thus may offer a promising therapeutic target for IPF.
Asunto(s)
Bleomicina/efectos adversos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fibrosis Pulmonar/inducido químicamente , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/farmacología , Bleomicina/administración & dosificación , Diferenciación Celular , Colágeno Tipo I/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Colágeno Tipo V/inmunología , Tolerancia Inmunológica/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/prevención & control , Adulto , Animales , Asma/sangre , Asma/inmunología , Asma/prevención & control , Colágeno Tipo V/sangre , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Hipersensibilidad Respiratoria/sangreRESUMEN
Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-ß mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-ßRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-ß mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-ß, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.