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Hypersensitivity pneumonitis is an immunologically mediated form of lung disease, resulting from inhalational exposure to a large variety of antigens. A subgroup of patients with fibrotic hypersensitivity pneumonitis (FHP) develop symptomatic, functional and radiographic disease progression. Mortality occurs primarily from respiratory failure as a result of progressive and self-sustaining lung injury that often occurs despite immunosuppression and removal of the inciting antigen. The development and validation of a prognostic transcriptomic signature for FHP (PREDICT-HP) is an observational multicentre cohort study designed to explore a transcriptomic signature from peripheral blood mononuclear cells in patients with FHP that is predictive of disease progression. This article describes the design and rationale of the PREDICT-HP study. This study will enrol â¼135 patients with FHP at approximately seven academic medical sites. Participants with a confirmed diagnosis of FHP are followed over 24â months and undergo physical examinations, self-administered questionnaires, chest computed tomography, pulmonary function tests, a 6-min walk test and blood testing for transcriptomic analyses. At each 6-month follow-up visit the study will assess the participants' clinical course and clinical events including hospitalisations and respiratory exacerbations. The PREDICT study has the potential to enhance our ability to predict disease progression and fundamentally advance our understanding of the pathobiology of FHP disease progression.
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Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.
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Estudo de Associação Genômica Ampla , Sarcoidose , Humanos , Predisposição Genética para Doença , Cadeias alfa de HLA-DR/genética , Leucócitos Mononucleares , Sarcoidose/genética , Cadeias HLA-DRB1/genética , AlelosRESUMO
BACKGROUND: Scientific discovery progresses by exploring new and uncharted territory. More specifically, it advances by a process of transforming unknown unknowns first into known unknowns, and then into knowns. Over the last few decades, researchers have developed many knowledge bases to capture and connect the knowns, which has enabled topic exploration and contextualization of experimental results. But recognizing the unknowns is also critical for finding the most pertinent questions and their answers. Prior work on known unknowns has sought to understand them, annotate them, and automate their identification. However, no knowledge-bases yet exist to capture these unknowns, and little work has focused on how scientists might use them to trace a given topic or experimental result in search of open questions and new avenues for exploration. We show here that a knowledge base of unknowns can be connected to ontologically grounded biomedical knowledge to accelerate research in the field of prenatal nutrition. RESULTS: We present the first ignorance-base, a knowledge-base created by combining classifiers to recognize ignorance statements (statements of missing or incomplete knowledge that imply a goal for knowledge) and biomedical concepts over the prenatal nutrition literature. This knowledge-base places biomedical concepts mentioned in the literature in context with the ignorance statements authors have made about them. Using our system, researchers interested in the topic of vitamin D and prenatal health were able to uncover three new avenues for exploration (immune system, respiratory system, and brain development) by searching for concepts enriched in ignorance statements. These were buried among the many standard enriched concepts. Additionally, we used the ignorance-base to enrich concepts connected to a gene list associated with vitamin D and spontaneous preterm birth and found an emerging topic of study (brain development) in an implied field (neuroscience). The researchers could look to the field of neuroscience for potential answers to the ignorance statements. CONCLUSION: Our goal is to help students, researchers, funders, and publishers better understand the state of our collective scientific ignorance (known unknowns) in order to help accelerate research through the continued illumination of and focus on the known unknowns and their respective goals for scientific knowledge.
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Bases de Conhecimento , Conhecimento , Processamento de Linguagem Natural , Feminino , Humanos , Recém-Nascido , Nascimento Prematuro , Publicações , Vitamina DRESUMO
The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.
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RNA , Transcriptoma , Citometria de Fluxo/métodos , Separação Celular , Padrões de ReferênciaRESUMO
BACKGROUND: The prevalence of atopic diseases has increased with atopic dermatitis (AD) as the earliest manifestation. We assessed if molecular risk factors in atopic mothers influence their infants' susceptibility to an atopic disease. METHODS: Pregnant women and their infants with (n = 174, high-risk) or without (n = 126, low-risk) parental atopy were enrolled in a prospective birth cohort. Global differentially methylated regions (DMRs) were determined in atopic (n = 92) and non-atopic (n = 82) mothers. Principal component analysis was used to predict atopy risk in children dependent on maternal atopy. Genome-wide transcriptomic analyses were performed in paired atopic (n = 20) and non-atopic (n = 15) mothers and cord blood. Integrative genomic analyses were conducted to define methylation-gene expression relationships. RESULTS: Atopic dermatitis was more prevalent in high-risk compared to low-risk children by age 2. Differential methylation analyses identified 165 DMRs distinguishing atopic from non-atopic mothers. Inclusion of DMRs in addition to maternal atopy significantly increased the odds ratio to develop AD in children from 2.56 to 4.26. In atopic compared to non-atopic mothers, 139 differentially expressed genes (DEGs) were identified significantly enriched of genes within the interferon signaling pathway. Expression quantitative trait methylation analyses dependent on maternal atopy identified 29 DEGs controlled by 136 trans-acting methylation marks, some located near transcription factors. Differential expression for the same nine genes, including MX1 and IFI6 within the interferon pathway, was identified in atopic and non-atopic mothers and high-risk and low-risk children. CONCLUSION: These data suggest that in utero epigenetic and transcriptomic mechanisms predominantly involving the interferon pathway may impact and predict the development of infant atopy.
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Dermatite Atópica , Criança , Lactente , Humanos , Feminino , Gravidez , Pré-Escolar , Dermatite Atópica/epidemiologia , Dermatite Atópica/genética , Estudos Prospectivos , Fatores de Risco , Família , TranscriptomaRESUMO
Multiple overlapping pathways are identified in tissue, BAL cells, PBMCs and a sarcoidosis in vitro granuloma model. Inferences from omic studies are constrained by small sample sizes. Studies comparing differences between sarcoidosis phenotypes are needed. https://bit.ly/30NaHz4.
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Amid the ongoing COVID-19 pandemic, it has become increasingly important to monitor the mutations that arise in the SARS-CoV-2 virus, to prepare public health strategies and guide the further development of vaccines and therapeutics. The spike (S) protein and the proteins comprising the RNA-Dependent RNA Polymerase (RdRP) are key vaccine and drug targets, respectively, making mutation surveillance of these proteins of great importance. Full protein sequences were downloaded from the GISAID database, aligned, and the variants identified. 437,006 unique viral genomes were analyzed. Polymorphisms in the protein sequence were investigated and examined longitudinally to identify sequence and strain variants appearing between January 5th, 2020 and January 16th, 2021. A structural analysis was also performed to investigate mutations in the receptor binding domain and the N-terminal domain of the spike protein. Within the spike protein, there were 766 unique mutations observed in the N-terminal domain and 360 in the receptor binding domain. Four residues that directly contact ACE2 were mutated in more than 100 sequences, including positions K417, Y453, S494, and N501. Within the furin cleavage site of the spike protein, a high degree of conservation was observed, but the P681H mutation was observed in 10.47% of sequences analyzed. Within the RNA dependent RNA polymerase complex proteins, 327 unique mutations were observed in Nsp8, 166 unique mutations were observed in Nsp7, and 1157 unique mutations were observed in Nsp12. Only 4 sequences analyzed contained mutations in the 9 residues that directly interact with the therapeutic Remdesivir, suggesting limited mutations in drug interacting residues. The identification of new variants emphasizes the need for further study on the effects of the mutations and the implications of increased prevalence, particularly for vaccine or therapeutic efficacy.
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COVID-19/epidemiologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , Genoma Viral , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , África/epidemiologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/farmacologia , Ásia/epidemiologia , Sítios de Ligação , COVID-19/virologia , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Bases de Dados Factuais , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Evolução Molecular , Furina/genética , Furina/metabolismo , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/classificação , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Estados Unidos/epidemiologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
The prognostic value of peripheral blood mononuclear cell (PBMC) expression profiles, when used in patients with chronic hypersensitivity pneumonitis (CHP), as an adjunct to traditional clinical assessment is unknown. RNA-seq analysis on PBMC from 37 patients with CHP at initial presentation determined that (1) 74 differentially expressed transcripts at a 10% false discovery rate distinguished those with (n=10) and without (n=27) disease progression, defined as absolute FVC and/or diffusing capacity of the lungs for carbon monoxide (DLCO) decline of ≥10% and increased fibrosis on chest CT images within 24 months, and (2) classification models based on gene expression and clinical factors strongly outperform models based solely on clinical factors (baseline FVC%, DLCO% and chest CT fibrosis).
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Alveolite Alérgica Extrínseca , Leucócitos Mononucleares , Alveolite Alérgica Extrínseca/diagnóstico por imagem , Alveolite Alérgica Extrínseca/genética , Humanos , Pulmão , Prognóstico , TranscriptomaRESUMO
INTRODUCTION: Sarcoidosis is a multiorgan granulomatous disorder thought to be triggered and influenced by gene-environment interactions. Sarcoidosis affects 45-300/100 000 individuals in the USA and has an increasing mortality rate. The greatest gap in knowledge about sarcoidosis pathobiology is a lack of understanding about the underlying immunological mechanisms driving progressive pulmonary disease. The objective of this study is to define the lung-specific and blood-specific longitudinal changes in the adaptive immune response and their relationship to progressive and non-progressive pulmonary outcomes in patients with recently diagnosed sarcoidosis. METHODS AND ANALYSIS: The BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints study is a US-based, NIH-sponsored longitudinal blood and bronchoscopy study. Enrolment will occur over four centres with a target sample size of 80 eligible participants within 18 months of tissue diagnosis. Participants will undergo six study visits over 18 months. In addition to serial measurement of lung function, symptom surveys and chest X-rays, participants will undergo collection of blood and two bronchoscopies with bronchoalveolar lavage separated by 6 months. Freshly processed samples will be stained and flow-sorted for isolation of CD4 +T helper (Th1, Th17.0 and Th17.1) and T regulatory cell immune populations, followed by next-generation RNA sequencing. We will construct bioinformatic tools using this gene expression to define sarcoidosis endotypes that associate with progressive and non-progressive pulmonary disease outcomes and validate the tools using an independent cohort. ETHICS AND DISSEMINATION: The study protocol has been approved by the Institutional Review Boards at National Jewish Hospital (IRB# HS-3118), University of Iowa (IRB# 201801750), Johns Hopkins University (IRB# 00149513) and University of California, San Francisco (IRB# 17-23432). All participants will be required to provide written informed consent. Findings will be disseminated via journal publications, scientific conferences, patient advocacy group online content and social media platforms.
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Sarcoidose Pulmonar , Sarcoidose , Líquido da Lavagem Broncoalveolar , Broncoscopia , Humanos , Estudos Multicêntricos como Assunto , Estudos Observacionais como Assunto , Linfócitos T Reguladores , Células Th17RESUMO
MOTIVATION: Science progresses by posing good questions, yet work in biomedical text mining has not focused on them much. We propose a novel idea for biomedical natural language processing: identifying and characterizing the questions stated in the biomedical literature. Formally, the task is to identify and characterize statements of ignorance, statements where scientific knowledge is missing or incomplete. The creation of such technology could have many significant impacts, from the training of PhD students to ranking publications and prioritizing funding based on particular questions of interest. The work presented here is intended as the first step towards these goals. RESULTS: We present a novel ignorance taxonomy driven by the role statements of ignorance play in research, identifying specific goals for future scientific knowledge. Using this taxonomy and reliable annotation guidelines (inter-annotator agreement above 80%), we created a gold standard ignorance corpus of 60 full-text documents from the prenatal nutrition literature with over 10 000 annotations and used it to train classifiers that achieved over 0.80 F1 scores. AVAILABILITY AND IMPLEMENTATION: Corpus and source code freely available for download at https://github.com/UCDenver-ccp/Ignorance-Question-Work. The source code is implemented in Python.
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Mast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage-determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes.
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Antígenos/metabolismo , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos/genética , Fator de Transcrição GATA2/genética , Mastócitos/metabolismo , Animais , Antígenos/imunologia , Linhagem da Célula/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Perfilação da Expressão Gênica/métodos , Masculino , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
The mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Although murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we have created a gene expression compendium across 24 distinct human and murine lung and LN MPs, along with human blood and murine spleen MPs, to serve as validation datasets. In-depth RNA sequencing identifies corresponding human-mouse MP subtypes and determines marker genes shared and divergent across species. Unexpectedly, only 13%-23% of the top 1,000 marker genes (i.e., genes not shared across species-specific MP subtypes) overlap in corresponding human-mouse MP counterparts. Lastly, CD88 in both species helps distinguish monocytes/macrophages from DCs. Our cross-species expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes or genes will prove fruitful.
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Perfilação da Expressão Gênica , Pulmão/citologia , Linfonodos/citologia , Fagócitos/metabolismo , Adulto , Animais , Antígenos CD1/metabolismo , Biomarcadores/metabolismo , Linhagem da Célula , Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , RNA/isolamento & purificação , Especificidade da EspécieRESUMO
More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.
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Quinase 9 Dependente de Ciclina/metabolismo , Histona Desmetilases/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Histona Desmetilases/química , Histona Desmetilases/genética , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , RNA Polimerase II/genéticaRESUMO
Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient's risk of severe COPD. We determined that 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in the immunoglobulin superfamily member 3 (IGSF3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs, of which rs1414272 and rs12066192 were directly and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin-enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide and sphingosine-1 phosphate but low glycosphingolipids and ganglioside levels, and they were less apoptotic and more adherent, with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
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Fumar Cigarros/genética , Predisposição Genética para Doença , Imunoglobulinas/genética , Proteínas de Membrana/genética , Doença Pulmonar Obstrutiva Crônica/genética , Translocação Genética/genética , Apoptose/genética , Adesão Celular/genética , Movimento Celular/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 4/genética , Fumar Cigarros/efeitos adversos , Feminino , Regulação da Expressão Gênica/genética , Humanos , Integrina beta1/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/patologia , Índice de Gravidade de Doença , Tetraspanina 29/genéticaRESUMO
Alveolar macrophages serve as central orchestrators of inflammatory responses in the lungs, both initiating their onset and promoting their resolution. However, the mechanisms that program macrophages for these dynamic responses are not fully understood. Over 95% of all mammalian genes undergo alternative pre-mRNA splicing. While alternative splicing has been shown to regulate inflammatory responses in macrophages in vitro, it has not been investigated on a genome-wide scale in vivo Here we used RNAseq to investigate alternative pre-mRNA splicing in alveolar macrophages isolated from lipopolysaccharide (LPS)-treated mice during the peak of inflammation and during its resolution. We found that lung inflammation induced substantial alternative pre-mRNA splicing in alveolar macrophages. The number of changes in isoform usage was greatest at the peak of inflammation and involved multiple classes of alternative pre-mRNA splicing events. Comparative pathway analysis of inflammation-induced changes in alternative pre-mRNA splicing and differential gene expression revealed overlap of pathways enriched for immune responses such as chemokine signaling and cellular metabolism. Moreover, alternative pre-mRNA splicing of genes in metabolic pathways differed in tissue resident vs. recruited (blood monocyte-derived) alveolar macrophages and corresponded to changes in core metabolism, including a switch to Warburg-like metabolism in recruited macrophages with increased glycolysis and decreased flux through the tricarboxylic acid cycle.
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Inflamação/genética , Macrófagos Alveolares/metabolismo , Precursores de RNA , Splicing de RNA , Animais , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos Endogâmicos C57BL , RNA-SeqRESUMO
Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.
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Linfócitos B/metabolismo , Linhagem da Célula/genética , DNA Helicases/genética , Linfopoese/genética , Transcrição Gênica/genética , Animais , Linfócitos B/citologia , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos TransgênicosRESUMO
The discovery of mutant tyrosine kinases as oncogenic drivers of lung adenocarcinomas has changed the basic understanding of lung cancer development and therapy. Yet, expressed kinases (kinome) in lung cancer progenitor cells, as well as whether kinase expression and the overall kinome changes or is reprogrammed upon transformation, is incompletely understood. We hypothesized that the kinome differs between lung cancer progenitor cells, alveolar type II cells (ATII), and basal cells (BC) and that their respective kinomes undergo distinct lineage-specific reprogramming to adenocarcinomas and squamous cell carcinomas upon transformation. We performed RNA sequencing on freshly isolated human ATII, BC, and lung cancer cell lines to define the kinome in nontransformed cells and transformed cells. Our studies identified a unique kinome for ATII and BC and changes in their kinome upon transformation to their respective carcinomas.
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Células-Tronco Adultas/enzimologia , Células Epiteliais Alveolares/enzimologia , Transformação Celular Neoplásica , Neoplasias Pulmonares/enzimologia , Pulmão/enzimologia , Proteínas de Neoplasias/análise , Proteínas Tirosina Quinases/análise , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Animais , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Linhagem da Célula , Células Cultivadas , Indução Enzimática , Humanos , Pulmão/citologia , Neoplasias Pulmonares/genética , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/enzimologia , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , RNA Mensageiro/análise , RNA Neoplásico/análise , TranscriptomaRESUMO
Autoreactive B cells have a major function in autoimmunity. A small subset of B cells expressing two distinct B-cell-antigen-receptors (B2R cells) is elevated in many patients with systematic lupus erythematosus (SLE) and in the MRL(/lpr) mouse model of lupus, and is often autoreactive. Here we show, using RNAseq and in vitro and in vivo analyses, signals that are required for promoting B2R cell numbers and effector function in autoimmune mice. Compared with conventional B cells, B2R cells are more responsive to Toll-like receptor 7/9 and type I/II interferon treatment, display higher levels of MHCII and co-receptors, and depend on IL-21 for their homeostasis; moreover they expand better upon T cell-dependent antigen stimulation, and mount a more robust memory response, which are characteristics essential for enhanced (auto)immune responses. Our findings thus provide insights on the stimuli for the expansion of an autoreactive B cell subset that may contribute to the etiology of SLE.
Assuntos
Autoanticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Lúpus Eritematoso Sistêmico/imunologia , Transdução de Sinais/imunologia , Imunidade Adaptativa/imunologia , Animais , Autoimunidade/imunologia , Subpopulações de Linfócitos B/metabolismo , Homeostase/imunologia , Imunidade Inata/imunologia , Interleucinas/imunologia , Interleucinas/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
RATIONALE: Cystic fibrosis pulmonary exacerbations accelerate pulmonary decline and increase mortality. Previously, we identified a 10-gene leukocyte panel measured directly from whole blood, which indicates response to exacerbation treatment. We hypothesized that molecular characteristics of exacerbations could also predict future disease severity. OBJECTIVES: We tested whether a 10-gene panel measured from whole blood could identify patient cohorts at increased risk for severe morbidity and mortality, beyond standard clinical measures. METHODS: Transcript abundance for the 10-gene panel was measured from whole blood at the beginning of exacerbation treatment (n = 57). A hierarchical cluster analysis of subjects based on their gene expression was performed, yielding four molecular clusters. An analysis of cluster membership and outcomes incorporating an independent cohort (n = 21) was completed to evaluate robustness of cluster partitioning of genes to predict severe morbidity and mortality. RESULTS: The four molecular clusters were analyzed for differences in forced expiratory volume in 1 second, C-reactive protein, return to baseline forced expiratory volume in 1 second after treatment, time to next exacerbation, and time to morbidity or mortality events (defined as lung transplant referral, lung transplant, intensive care unit admission for respiratory insufficiency, or death). Clustering based on gene expression discriminated between patient groups with significant differences in forced expiratory volume in 1 second, admission frequency, and overall morbidity and mortality. At 5 years, all subjects in cluster 1 (very low risk) were alive and well, whereas 90% of subjects in cluster 4 (high risk) had suffered a major event (P = 0.0001). In multivariable analysis, the ability of gene expression to predict clinical outcomes remained significant, despite adjustment for forced expiratory volume in 1 second, sex, and admission frequency. The robustness of gene clustering to categorize patients appropriately in terms of clinical characteristics, and short- and long-term clinical outcomes, remained consistent, even when adding in a secondary population with significantly different clinical outcomes. CONCLUSIONS: Whole blood gene expression profiling allows molecular classification of acute pulmonary exacerbations, beyond standard clinical measures, providing a predictive tool for identifying subjects at increased risk for mortality and disease progression.
Assuntos
Proteína C-Reativa/genética , Fibrose Cística/genética , Perfilação da Expressão Gênica , Adulto , Biomarcadores/sangue , Colúmbia Britânica/epidemiologia , Proteína C-Reativa/metabolismo , Colorado/epidemiologia , Fibrose Cística/diagnóstico , Fibrose Cística/epidemiologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Morbidade/tendências , Prognóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Taxa de Sobrevida/tendências , Fatores de TempoRESUMO
Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-Fhi resident alveolar MΦ and a mixed population of CD11bhi MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP) to CD11bhi MΦ. Upon loss of c-FLIP, CD11bhi MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11bhi MΦ, but not Siglec-Fhi MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.