RESUMO
Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, Pâ=â1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have â¼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (Pâ=â10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/genética , Avaliação Pré-Clínica de Medicamentos , Alelos , Animais , Antígenos CD19/genética , Artrite Reumatoide/patologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Locos de Características Quantitativas/genética , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
OBJECTIVE: The significance of non-rheumatoid arthritis (RA) autoantibodies in patients with RA is unclear. The aim of this study was to assess associations of autoantibodies with autoimmune risk alleles and with clinical diagnoses from the electronic medical records (EMRs) among RA cases and non-RA controls. METHODS: Data on 1,290 RA cases and 1,236 non-RA controls of European genetic ancestry were obtained from the EMRs of 2 large academic centers. The levels of anti-citrullinated protein antibodies (ACPAs), antinuclear antibodies (ANAs), anti-tissue transglutaminase antibodies (AGTAs), and anti-thyroid peroxidase (anti-TPO) antibodies were measured. All subjects were genotyped for autoimmune risk alleles, and the association between number of autoimmune risk alleles present and number of types of autoantibodies present was studied. A phenome-wide association study (PheWAS) was conducted to study potential associations between autoantibodies and clinical diagnoses among RA cases and non-RA controls. RESULTS: The mean ages were 60.7 years in RA cases and 64.6 years in non-RA controls. The proportion of female subjects was 79% in each group. The prevalence of ACPAs and ANAs was higher in RA cases compared to controls (each P < 0.0001); there were no differences in the prevalence of anti-TPO antibodies and AGTAs. Carriage of higher numbers of autoimmune risk alleles was associated with increasing numbers of autoantibody types in RA cases (P = 2.1 × 10(-5)) and non-RA controls (P = 5.0 × 10(-3)). From the PheWAS, the presence of ANAs was significantly associated with a diagnosis of Sjögren's/sicca syndrome in RA cases. CONCLUSION: The increased frequency of autoantibodies in RA cases and non-RA controls was associated with the number of autoimmune risk alleles carried by an individual. PheWAS of EMR data, with linkage to laboratory data obtained from blood samples, provide a novel method to test for the clinical significance of biomarkers in disease.
Assuntos
Anticorpos Antinucleares/sangue , Artrite Reumatoide , Autoanticorpos/sangue , Hipotireoidismo , Síndrome de Sjogren , Idoso , Anticorpos Antinucleares/genética , Artrite Reumatoide/epidemiologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Autoanticorpos/genética , Registros Eletrônicos de Saúde , Feminino , Proteínas de Ligação ao GTP/imunologia , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Hipotireoidismo/epidemiologia , Hipotireoidismo/genética , Hipotireoidismo/imunologia , Iodeto Peroxidase/imunologia , Masculino , Pessoa de Meia-Idade , Peptídeos Cíclicos/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de Risco , Estudos Soroepidemiológicos , Síndrome de Sjogren/epidemiologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Tireoidite/epidemiologia , Tireoidite/genética , Tireoidite/imunologia , Transglutaminases/imunologia , População Branca/genética , População Branca/estatística & dados numéricosRESUMO
RATIONALE: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. OBJECTIVES: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. METHODS: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. MEASUREMENTS AND MAIN RESULTS: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. CONCLUSIONS: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.
Assuntos
Fator 4 Ativador da Transcrição/genética , Brônquios/metabolismo , Células Epiteliais/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Transcriptoma/fisiologia , Idoso , Análise de Variância , Androstadienos , Brônquios/efeitos dos fármacos , Broncodilatadores/farmacologia , Broncoscopia , Células Epiteliais/efeitos dos fármacos , Feminino , Fluticasona , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Testes de Função Respiratória , Transcriptoma/efeitos dos fármacosRESUMO
SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments have not been fully characterized using matched samples from large cohorts. Gene expression data from 793 nasal and 1673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking status (current versus former) was the only clinical factor significantly and reproducibly associated with VE gene expression. The expression of ACE2 and TMPRSS2 was higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the gene expression data confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose, these genes are not predominantly expressed in cell populations modulated by smoking. In individuals with elevated lung cancer risk, smoking-induced VE gene expression changes in the nose likely have minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.
Assuntos
COVID-19 , Neoplasias Pulmonares , Humanos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Detecção Precoce de Câncer , Peptidil Dipeptidase A/metabolismo , Neoplasias Pulmonares/metabolismo , Brônquios/metabolismo , Fumar/efeitos adversos , Fumar/genéticaRESUMO
Background : SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments has not been fully characterized using matched samples from large cohorts. Results : Gene expression data from 793 nasal and 1,673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking was the only clinical factor significantly and reproducibly associated with VE gene expression. ACE2 and TMPRSS2 expression were higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48 + secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the RNA-seq confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. Conclusions : The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose these genes are not predominantly expressed in cell populations modulated by smoking. Smoking-induced VE gene expression changes in the nose likely has minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.
RESUMO
Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.
Assuntos
COVID-19/epidemiologia , COVID-19/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/fisiologia , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Internalização do Vírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , COVID-19/virologia , Catepsina L/genética , Catepsina L/metabolismo , Conjuntos de Dados como Assunto/estatística & dados numéricos , Demografia , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Análise de Sequência de RNA/métodos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única/métodosRESUMO
Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Broncogênico/patologia , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias Pulmonares/patologia , Lesões Pré-Cancerosas/patologia , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/imunologia , Biópsia , Brônquios/diagnóstico por imagem , Brônquios/imunologia , Brônquios/patologia , Broncoscopia , Carcinoma Broncogênico/genética , Carcinoma Broncogênico/imunologia , Carcinoma Broncogênico/prevenção & controle , Estudos de Coortes , Conjuntos de Dados como Assunto , Progressão da Doença , Detecção Precoce de Câncer/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/diagnóstico por imagem , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/imunologia , RNA Mensageiro/genética , Mucosa Respiratória/citologia , Mucosa Respiratória/diagnóstico por imagem , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Análise de Sequência de RNA , Linfócitos T/imunologia , Tomografia Computadorizada por Raios X , Regulação para CimaRESUMO
The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.