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1.
Viruses ; 13(3)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799525

RESUMO

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4's acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

2.
Front Immunol ; 12: 633654, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33732255

RESUMO

Epigenetics plays an important role in the priming the dynamic response of airway epithelial cells to infectious and environmental stressors. Here, we examine the epigenetic role of the SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin A4 (SMARCA4) in the epithelial response to RSV infection. Depletion of SMARCA4 destabilized the abundance of the SMARCE1/ARID1A SWI/SNF subunits, disrupting the innate response and triggering a hybrid epithelial/mesenchymal (E/M) state. Assaying SMARCA4 complex-regulated open chromatin domains by transposase cleavage -next generation sequencing (ATAC-Seq), we observed that the majority of cleavage sites in uninfected cells have reduced chromatin accessibility. Paradoxically, SMARCA4 complex-depleted cells showed enhanced RSV-inducible chromatin opening and gene expression in the EMT pathway genes, MMP9, SNAI1/2, VIM, and CDH2. Focusing on the key MMP9, we observed that SMARCA4 complex depletion reduced basal BRD4 and RNA Polymerase II binding, but enhanced BRD4/Pol II binding in response to RSV infection. In addition, we observed that MMP9 secretion in SMARCA4 complex deficient cells contributes to mesenchymal transition, cellular fusion (syncytia) and subepithelial myofibroblast transition. We conclude the SMARCA4 complex is a transcriptional repressor of epithelial plasticity, whose depletion triggers a hybrid E/M state that affects the dynamic response of the small airway epithelial cell in mucosal remodeling via paracrine MMP9 activity.

3.
Viruses ; 13(2)2021 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-33572560

RESUMO

The innate immune response (IIR) involves rapid genomic expression of protective interferons (IFNs) and inflammatory cytokines triggered by intracellular viral replication. Although the transcriptional control of the innate pathway is known in substantial detail, little is understood about the complexity of alternative splicing (AS) and alternative polyadenylation (APA) of mRNAs underlying the cellular IIR. In this study, we applied single-molecule, real-time (SMRT) sequencing with mRNA quantitation using short-read mRNA sequencing to characterize changes in mRNA processing in the epithelial response to respiratory syncytial virus (RSV) replication. Mock or RSV-infected human small-airway epithelial cells (hSAECs) were profiled using SMRT sequencing and the curated transcriptome analyzed by structural and quality annotation of novel transcript isoforms (SQANTI). We identified 113,082 unique isoforms; 28,561 represented full splice matches, and 45% of genes expressed six or greater AS mRNA isoforms. Identification of differentially expressed AS isoforms was accomplished by mapping a short-read RNA sequencing expression matrix to the curated transcriptome, and 905 transcripts underwent differential polyadenylation site analysis enriched in protein secretion, translation, and mRNA degradation. We focused on 355 genes showing differential isoform utilization (DIU), indicating where a new AS isoform becomes a major fraction of mRNA isoforms expressed. In pathway and network enrichment analyses, we observed that DIU transcripts are substantially enriched in cell cycle control and IIR pathways. Interestingly, the RelA/IRF7 innate regulators showed substantial DIU where major transcripts included distinct isoforms with exon occlusion, intron inclusion, and alternative transcription start site utilization. We validated the presence of RelA and IRF7 AS isoforms as well as their induction by RSV using eight isoform-specific RT-PCR assays. These isoforms were identified in both immortalized and primary small-airway epithelial cells. We concluded that the cell cycle and IIR are differentially spliced in response to RSV. These data indicate that substantial post-transcriptional complexity regulates the antiviral response.


Assuntos
Processamento Alternativo , Imunidade Inata , RNA Mensageiro/genética , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/fisiologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Humanos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia
4.
J Proteome Res ; 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33054244

RESUMO

Metabolomics-the endpoint of the omics cascade-is increasingly recognized as a preferred method for understanding the ultimate responses of biological systems to stress. Flow injection electrospray (FIE) mass spectrometry (MS) has advantages for untargeted metabolic fingerprinting due to its simplicity and capability for high-throughput screening but requires a high-resolution mass spectrometer to resolve metabolite features. In this study, we developed and validated a high-throughput and highly reproducible metabolomics platform integrating FIE with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS for analysis of both polar and nonpolar metabolite features from plasma samples. FIE-FTICR MS enables high-throughput detection of hundreds of metabolite features in a single mass spectrum without a front-end separation step. Using plasma samples from genetically identical obese mice with or without type 2 diabetes (T2D), we validated the intra and intersample reproducibility of our method and its robustness for simultaneously detecting alterations in both polar and nonpolar metabolite features. Only 5 min is needed to acquire an ultra-high resolution mass spectrum in either a positive or negative ionization mode. Approximately 1000 metabolic features were reproducibly detected and annotated in each mouse plasma group. For significantly altered and highly abundant metabolite features, targeted tandem MS (MS/MS) analyses can be applied to confirm their identity. With this integrated platform, we successfully detected over 300 statistically significant metabolic features in T2D mouse plasma as compared to controls and identified new T2D biomarker candidates. This FIE-FTICR MS-based method is of high throughput and highly reproducible with great promise for metabolomics studies toward a better understanding and diagnosis of human diseases.

5.
Viruses ; 12(8)2020 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-32722537

RESUMO

Lower respiratory tract infection (LRTI) with respiratory syncytial virus (RSV) is associated with reduced lung function through unclear mechanisms. In this study, we test the hypothesis that RSV infection induces genomic reprogramming of extracellular matrix remodeling pathways. For this purpose, we sought to identify transcriptionally active open chromatin domains using assay for transposase-accessible-next generation sequencing (ATAC-Seq) in highly differentiated lower airway epithelial cells. High confidence nucleosome-free regions were those predicted independently using two peak-calling algorithms. In uninfected cells, ~12,650 high-confidence open chromatin regions were identified. These mapped to ~8700 gene bodies, whose genes functionally controlled organelle synthesis and Th2 pathways (IL6, TSLP). These latter cytokines are preferentially secreted by RSV-infected bronchiolar cells and linked to mucous production, obstruction, and atopy. By contrast, in RSV infection, we identify ~1700 high confidence open chromatin domains formed in 1120 genes, primarily in introns. These induced chromatin modifications are associated with complex gene expression profiles controlling tyrosine kinase growth factor signaling and extracellular matrix (ECM) secretory pathways. Of these, RSV induces formation of nucleosome-free regions on TGFB1/JUNB//FN1/MMP9 genes and the rate limiting enzyme in the hexosamine biosynthetic pathway (HBP), Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). RSV-induced open chromatin domains are highly enriched in AP1 binding motifs and overlap experimentally determined JUN peaks in GEO ChIP-Seq data sets. Our results provide a topographical map of chromatin accessibility and suggest a growth factor and AP1-dependent mechanism for upregulation of the HBP and ECM remodeling in lower epithelial cells that may be linked to long-term airway remodeling.

6.
Viruses ; 12(4)2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32331282

RESUMO

Respiratory syncytial virus infection is responsible for seasonal upper and lower respiratory tract infections worldwide, causing substantial morbidity. Self-inoculation of the virus into the nasopharynx results in epithelial replication and distal spread into the lower respiratory tract. Here, respiratory syncytial virus (RSV) activates sentinel cells important in the host inflammatory response, resulting in epithelial-derived cytokine and interferon (IFN) expression resulting in neutrophilia, whose intensity is associated with disease severity. I will synthesize key findings describing how RSV replication activates intracellular NFκB and IRF signaling cascades controlling the innate immune response (IIR). Recent studies have implicated a central role for Scg1a1+ expressing progenitor cells in IIR, a cell type uniquely primed to induce neutrophilic-, T helper 2 (Th2)-polarizing-, and fibrogenic cytokines that play distinct roles in disease pathogenesis. Molecular studies have linked the positive transcriptional elongation factor-b (P-TEFb), a pleiotrophic chromatin remodeling complex in immediate-early IIR gene expression. Through intrinsic kinase activity of cyclin dependent kinase (CDK) 9 and atypical histone acetyl transferase activity of bromodomain containing protein 4 (BRD4), P-TEFb mediates transcriptional elongation of IIR genes. Unbiased proteomic studies show that the CDK9•BRD4 complex is dynamically reconfigured by the innate response and targets TGFß-dependent fibrogenic gene networks. Chronic activation of CDK9•BRD4 mediates chromatin remodeling fibrogenic gene networks that cause epithelial mesenchymal transition (EMT). Mesenchymal transitioned epithelial cells elaborate TGFß and IL6 that function in a paracrine manner to expand the population of subepithelial myofibroblasts. These findings may account for the long-term reduction in pulmonary function in children with severe lower respiratory tract infection (LRTI). Modifying chromatin remodeling properties of the CDK9•BRD4 coactivators may provide a mechanism for reducing post-infectious airway remodeling that are a consequence of severe RSV LRTIs.

7.
J Med Chem ; 63(10): 5242-5256, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32255647

RESUMO

Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Cromonas/administração & dosagem , Cromonas/química , Descoberta de Drogas/métodos , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromonas/farmacologia , Cristalização/métodos , Cristalização/tendências , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
8.
Am J Pathol ; 190(2): 306-322, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31955791

RESUMO

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conorii-infected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses.


Assuntos
Biomarcadores/sangue , Febre Botonosa/diagnóstico , Células Endoteliais da Veia Umbilical Humana/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/sangue , Proteoma/análise , Infecções por Rickettsia/complicações , Rickettsia/patogenicidade , Animais , Febre Botonosa/epidemiologia , Febre Botonosa/microbiologia , Estudos de Coortes , Feminino , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteômica , Rickettsia/isolamento & purificação , Infecções por Rickettsia/microbiologia , Infecções por Rickettsia/transmissão , Texas/epidemiologia
9.
Drug Discov Today ; 25(1): 126-132, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733396

RESUMO

Structural remodeling is central to the initiation and progression of many chronic lung diseases, representing an important unmet need. We examine the evidence supporting bromodomain-containing protein 4 (BRD4) as a validated biological target for treatment of airway remodeling. In epithelial cells and fibroblasts, BRD4 serves as a scaffold for chromatin remodeling complexes in active super-enhancers. In response to inflammatory stimuli, BRD4 is repositioned to innate and mesenchymal genes activating their production. Proof-of-concept studies show promising benefit of selective BRD4 inhibitors in disrupting epithelial mesenchymal transition and myofibroblast transition in diverse models of lung injury. Recent identification of biomarkers of BRD4 provides a basis for further drug development for application in viral-induced airway inflammation, COPD and interstitial lung diseases.

11.
Artigo em Inglês | MEDLINE | ID: mdl-31692901

RESUMO

Allergic asthma is a chronic inflammatory airway disease whose clinical course is punctuated by acute exacerbations from aeroallergen exposure or respiratory virus infections. Aeroallergens and respiratory viruses stimulate toll-like receptor (TLR) signaling, producing oxidative injury and inflammation. Repetitive exacerbations produce complex mucosal adaptations, cell-state changes, and structural remodeling. These structural changes produce substantial morbidity, decrease lung capacity, and impair quality of life. We will review recent systems-level studies that provide fundamental new insights into how repetitive activation of innate signaling pathways produce epigenetic 'training' to induce adaptive epithelial responses. Oxidative stress produced downstream of TLR signaling induces transient oxidation of guanine bases in the regulatory regions of inflammatory genes. The epigenetic mark 8-oxoG is bound by a pleiotropic DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), which induces conformational changes in adjacent DNA to recruit the NFκB·bromodomain-containing protein 4 (BRD4) complex. The NFκB·BRD4 complex not only plays a central role in inflammation, but also triggers mesenchymal transition and extracellular matrix remodeling. Small molecule inhibitors of OGG1-8-oxoG binding and BRD4-acetylated histone interaction have been developed. We present studies demonstrating efficacy of these in reducing airway inflammation in preclinical models. Targeting inducible epigenetic reprogramming pathway shows promise for therapeutics in reversing airway remodeling in a variety of chronic airway diseases.

12.
J Proteome Res ; 18(9): 3447-3460, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31424945

RESUMO

Type II epithelial-mesenchymal transition (EMT) plays a vital role in airway injury, repair, and remodeling. Triggered by growth factors, such as transforming growth factor beta (TGFß), EMT induced a biological process that converts epithelial cells into secretory mesenchymal cells with a substantially increased production of extracellular matrix (ECM) proteins. Epithelial cells are not professional secretory cells and produce few ECM proteins under normal conditions. The molecular mechanism underlying the transformation of the protein factory and secretory machinery during EMT is significant because ECM secretion is central to the pathogenesis of airway remodeling. Here we report that type II EMT upregulates the protein N-glycosylation of ECMs. The mechanism study reveals that the substantial increase in synthesis of ECM proteins in EMT activates the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 (XBP1) axis of the unfolded protein response (UPR) coupled to the hexosamine biosynthesis pathway (HBP). These two pathways coordinately up-regulate the protein N-glycosylation of ECM proteins and increase ER folding capacity and ER-associated degradation (ERAD), which improve ER protein homeostasis and protect transitioned cells from proteotoxicity. Inhibition of the alternative splicing of XBP1 or protein N-glycosylation blocks ECM protein secretion, indicating the XBP1-HBP plays a prominent role in regulating the secretion of ECM proteins in the mesenchymal transition. Our data suggest that the activation of XBP1-HBP pathways and elevation of protein N-glycosylation is an adaptive response to maintain protein quality control and facilitate the secretion of ECM proteins during the mesenchymal transition. The components of the XBP1-HBP pathways may be therapeutic targets to prevent airway remodeling.


Assuntos
Remodelação das Vias Aéreas/genética , Endorribonucleases/genética , Lesão Pulmonar/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Glicosilação , Hexosaminas/genética , Hexosaminas/metabolismo , Humanos , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Proteostase/genética , Transdução de Sinais/genética
13.
J Proteomics ; 205: 103415, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31195152

RESUMO

Small molecule inhibitors of the epigenetic regulator bromodomain-containing protein 4 (BRD4) are potential therapeutics for viral and allergen-induced airway remodeling. A limitation of their preclinical advancement is the lack of detailed understanding of mechanisms of action and biomarkers of effect. We report a systems-level pharmacoproteomics in a standardized murine model of toll-like receptor TLR3-NFκB/RelA innate inflammation in the absence or presence of a highly selective BRD4 inhibitor (ZL0454) or nonselective bromodomain and extraterminal domain inhibitor (JQ1). Proteomics of bronchoalveolar lavage fluid (BALF) secretome and exosomal proteins from this murine model revealed increased, selective, capillary leak associated with pericyte-myofibroblast transition, a phenomenon blocked by BRD4 inhibitors. BALF proteomics also suggested that ZL0454 better reduced the vascular leakage and extracellular matrix deposition than JQ1. A significant subset of inflammation-mediated remodeling factors was also identified in a mouse model of idiopathic pulmonary fibrosis produced by bleomycin. BALF exosome analysis indicated that BRD4 inhibitors reduced the induction of exosomes enriched in coagulation factors whose presence correlated with interstitial fibrin deposition. Finally, BALF samples from humans with severe asthma demonstrated similar upregulations of ORM2, APCS, SPARCL1, FGA, and FN1, suggesting their potential as biomarkers for early detection of airway remodeling and/or monitoring of therapy response. SIGNIFICANCE: Repetitive and chronic viral upper respiratory tract infections trigger toll-like receptor (TLR)3-NFκB/RelA mediated airway remodeling which is linked to a progressive decline in pulmonary function in patients with asthma and chronic obstructive pulmonary disease. Small molecule inhibitors of the epigenetic regulator bromodomain-containing protein 4 (BRD4) are potential therapeutics for viral and allergen-induced airway remodeling. A limitation of their preclinical advancement is the lack of detailed understanding of mechanisms of action and biomarkers of effect. Our study revealed that the activation of (TLR)3-NFκB/RelA pathway in the lung induced an elevation in coagulation, complement, and platelet factors, indicating the increased vascular leak during airway remodeling. The mechanism of vascular leakage was chronic inflammation-induced pericyte-myofibroblast transition, which was blocked by BRD4 inhibitors. Finally, proteomics analysis of the bronchoalveolar lavage fluid samples from humans with severe asthma demonstrated similar findings that we observed in the animal model.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Biomarcadores Farmacológicos/análise , Vasos Sanguíneos/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Citoproteção/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Animais , Asma/metabolismo , Asma/patologia , Azepinas/farmacologia , Biomarcadores Farmacológicos/metabolismo , Bleomicina , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poli I-C/farmacologia , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Sulfonamidas/farmacologia , Receptor 3 Toll-Like/fisiologia , Triazóis/farmacologia
14.
Expert Rev Respir Med ; 13(4): 349-356, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30712413

RESUMO

INTRODUCTION: Activation of antigen-independent inflammation (a.k.a. the 'innate' immune response (IIR)) plays a complex role in allergic asthma (AA). Although activation of the pulmonary IIR by aerosolized bacterial lipopolysaccharide early in life may be protective of AA, respiratory viral infections promote AA. The mechanisms how the mucosal IIR promotes allergic sensitization, remodeling, and altered epithelial signaling are not understood. Areas covered: This manuscript overviews: 1. Mechanistic studies identifying how allergens and viral patterns activate the mucosal IIR; 2. Research that reveals a major role played by specialized epithelial cells in the bronchiolar-alveolar junction in triggering inflammation and remodeling; 3. Reports linking the mucosal IIR with epithelial cell-state change and barrier disruption; and, 4. Observations relating mesenchymal transition with the expansion of the myofibroblast population. Expert commentary: Luminal allergens and viruses activate TLR signaling in key sentinel cells producing epithelial cell state transition, disrupting epithelial barrier function, and expanding the pulmonary myofibroblast population. These signals are transduced through a common NFκB/RelA -bromodomain containing four (BRD4) pathway, an epigenetic remodeling complex reprogramming the genome. Through this pathway, the mucosal IIR is a major modifier of adaptive immunity, AA and acute exacerbation-induced remodeling.


Assuntos
Asma/imunologia , Hipersensibilidade/imunologia , Imunidade Inata , Inflamação , Mucosa Respiratória/imunologia , Animais , Asma/fisiopatologia , Epigenômica , Humanos , Hipersensibilidade/fisiopatologia , Mucosa Respiratória/fisiopatologia
15.
J Allergy Clin Immunol ; 143(4): 1380-1394.e9, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30321559

RESUMO

BACKGROUND: Frequent exacerbations of allergic asthma lead to airway remodeling and a decrease in pulmonary function, producing morbidity. Cat dander is an aeroallergen associated with asthma risk. OBJECTIVE: We sought to elucidate the mechanism of cat dander-induced inflammation-remodeling. METHODS: We identified remodeling in mucosal samples from allergic asthma by using quantitative RT-PCR. We developed a model of aeroallergen-induced experimental asthma using repetitive cat dander extract exposure. We measured airway inflammation using immunofluorescence, leukocyte recruitment, and quantitative RT-PCR. Airway remodeling was measured by using histology, collagen content, myofibroblast numbers, and selected reaction monitoring. Inducible nuclear factor κB (NF-κB)-BRD4 interaction was measured by using a proximity ligation assay in situ. RESULTS: Enhanced mesenchymal signatures are observed in bronchial biopsy specimens from patients with allergic asthma. Cat dander induces innate inflammation through NF-κB signaling, followed by production of a profibrogenic mesenchymal transition in primary human small airway epithelial cells. The IκB kinase-NF-κB signaling pathway is required for mucosal inflammation-coupled airway remodeling and myofibroblast expansion in the mouse model of aeroallergen exposure. Cat dander induces NF-κB/RelA to complex with and activate BRD4, resulting in modifying the chromatin environment of inflammatory and fibrogenic genes through its atypical histone acetyltransferase activity. A novel small-molecule BRD4 inhibitor (ZL0454) disrupts BRD4 binding to the NF-κB-RNA polymerase II complex and inhibits its histone acetyltransferase activity. ZL0454 prevents epithelial mesenchymal transition, myofibroblast expansion, IgE sensitization, and fibrosis in airways of naive mice exposed to cat dander. CONCLUSIONS: NF-κB-inducible BRD4 activity mediates cat dander-induced inflammation and remodeling. Therapeutic modulation of the NF-κB-BRD4 pathway affects allergen-induced inflammation, epithelial cell-state changes, extracellular matrix production, and expansion of the subepithelial myofibroblast population.


Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/patologia , Proteínas de Ciclo Celular/metabolismo , Inflamação/imunologia , Mucosa Respiratória/patologia , Fatores de Transcrição/metabolismo , Animais , Asma/imunologia , Asma/metabolismo , Gatos , Alérgenos Animais/imunologia , Transição Epitelial-Mesenquimal/imunologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo
16.
Am J Respir Cell Mol Biol ; 60(1): 68-83, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30153047

RESUMO

NF-κB/RelA triggers innate inflammation by binding to bromodomain-containing protein 4 (BRD4), an atypical histone acetyltransferase (HAT). Although RelA·BRD4 HAT mediates acute neutrophilic inflammation, its role in chronic and functional airway remodeling is not known. We observed that BRD4 is required for Toll-like receptor 3 (TLR3)-mediated mesenchymal transition, a cell-state change that is characteristic of remodeling. We therefore tested two novel highly selective BRD4 inhibitors, ZL0420 and ZL0454, for their effects on chronic airway remodeling produced by repetitive TLR3 agonist challenges, and compared their efficacy with that of two nonselective bromodomain and extraterminal (BET) protein inhibitors, JQ1 and RVX208. We observed that ZL0420 and ZL0454 more potently reduced polyinosinic:polycytidylic acid-induced weight loss and fibrosis as assessed by microcomputed tomography and second harmonic generation microscopy. These measures correlated with the collagen deposition observed in histopathology. Importantly, the ZL inhibitors were more effective than the nonselective BET inhibitors at equivalent doses. The ZL inhibitors had significant effects on lung physiology, reversing TLR3-associated airway hyperresponsiveness and increasing lung compliance in vivo. At the molecular level, ZL inhibitors reduced elaboration of the transforming growth factor-ß-induced growth program, thereby preventing mucosal mesenchymal transition and disrupting BRD4 HAT activity and complex formation with RelA. We also observed that ZL0454 treatment blocked polyinosinic:polycytidylic acid-associated expansion of the α-SMA1+/COL1A+ myofibroblast population and prevented myofibroblast transition in a coculture system. We conclude that 1) BRD4 is a central effector of the mesenchymal transition that results in paracrine activation of myofibroblasts, mechanistically linking innate inflammation to airway hyperresponsiveness and fibrosis, and 2) highly selective BRD4 inhibitors may be effective in reversing the effects of repetitive airway viral infections on innate inflammation-mediated remodeling.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Inflamação/fisiopatologia , Proteínas Nucleares/antagonistas & inibidores , Fibrose Pulmonar/tratamento farmacológico , Mucosa Respiratória/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Remodelação das Vias Aéreas/fisiologia , Animais , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Humanos , Imunidade Inata/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , RNA Interferente Pequeno/genética , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo
17.
Front Immunol ; 9: 2413, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405613

RESUMO

APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (ß2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole ß2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.


Assuntos
Síndrome Antifosfolipídica/etiologia , Síndrome Antifosfolipídica/metabolismo , Coagulação Sanguínea , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Domínios e Motivos de Interação entre Proteínas , beta 2-Glicoproteína I/metabolismo , Adulto , Animais , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/diagnóstico , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica , Domínios Proteicos , Trombose/sangue , Trombose/etiologia , Trombose/metabolismo , beta 2-Glicoproteína I/química
18.
J Immunol ; 201(9): 2753-2766, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275049

RESUMO

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm and is subsequently released to the extracellular space during infection and injury. Once released, it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells and measured the release of proinflammatory mediators and the activation of NF-κB and P38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and P38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and P38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.


Assuntos
Proteína HMGB1/imunologia , Leucócitos Mononucleares/imunologia , Mucosa Respiratória/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Humanos , Imunidade Inata/imunologia , Vírus Sincicial Respiratório Humano/imunologia
19.
Expert Rev Respir Med ; 12(11): 931-939, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30241450

RESUMO

INTRODUCTION: Acute exacerbations of chronic lung disease account for substantial morbidity and health costs. Repeated inflammatory episodes and attendant bronchoconstriction cause structural remodeling of the airway. Remodeling is a multicellular response to mucosal injury that results in epithelial cell-state changes, enhanced extracellular deposition, and expansion of pro-fibrotic myofibroblast populations. Areas covered: This manuscript overviews mechanistic studies identifying key sentinel cell populations in the airway and how pattern recognition signaling induces maladaptive mucosal changes and airway remodeling. Studies elucidating how NFκB couples with an atypical histone acetyltransferase, bromodomain-containing protein 4 (BRD4) that reprograms mucosal fibrogenic responses, are described. The approaches to development and characterization of selective inhibitors of epigenetic reprogramming on innate inflammation and structural remodeling in preclinical models are detailed. Expert commentary: Bronchiolar cells derived from Scgb1a1-expressing progenitors function as major sentinel cells of the airway, responsible for initiating antiviral and aeroallergen responses. In these sentinel cells, activation of innate inflammation is coupled to neutrophilic recruitment, mesenchymal transition and myofibroblast expansion. Therapeutics targeting the NFkB-BRD4 may be efficacious in reducing pathological effects of acute exacerbations in chronic lung disease.


Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Imunidade Adaptativa/fisiologia , Alérgenos/imunologia , Hiper-Reatividade Brônquica/imunologia , Bronquíolos/citologia , Proteínas de Ciclo Celular , Exposição Ambiental/efeitos adversos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Imunidade Inata/fisiologia , Miofibroblastos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/metabolismo , Mucosa Respiratória/imunologia , Receptores Toll-Like/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Viroses/imunologia
20.
Sci Rep ; 8(1): 13348, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190498

RESUMO

Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.


Assuntos
Matriz Extracelular/patologia , Imageamento Tridimensional , Pulmão/patologia , Fibrose Pulmonar/patologia , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Poli I-C/efeitos adversos , Poli I-C/farmacologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo
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