RESUMO
BACKGROUND: Elevated levels of extracellular adenosine triphosphate (ATP) modulate immunologic pathways and are considered to be a danger signal in inflammation, lung fibrosis and cancer. Macrophages can be classified into two main types: M1 macrophages are classically activated, pro-inflammatory macrophages, whereas M2 macrophages are alternatively activated, pro-fibrotic macrophages. In this study, we examined the effect of ATP on differentiation of native human monocytes into these macrophage subtypes. We characterized M1 and M2 like macrophages by their release of Interleukin-1beta (IL-1ß) and Chemokine (C-C motif) ligand 18 (CCL18), respectively. RESULTS: Monocytes were stimulated with ATP or the P2X7 receptor agonist Benzoylbenzoyl-ATP (Bz-ATP), and the production of various cytokines was analyzed, with a particular focus on CCL18 and IL-1ß, along with the expression of different purinergic receptors. Over a 72 h period of cell culture, monocytes spontaneously differentiated to M2 like macrophages, as indicated by an increased release of CCL18. Immediate stimulation of monocytes with ATP resulted in a dose-dependent reduction in CCL18 release, but had no effect on the concentration of IL-1ß. In contrast, delayed stimulation with ATP had no effect on either CCL18 or IL-1ß release. Similar results were observed in a model of inflammation using lipopolysaccharide-stimulated human monocytes. Stimulation with the P2X7 receptor agonist Bz-ATP mimicked the effect of ATP on M2-macrophage differentiation, indicating that P2X7 is involved in ATP-induced inhibition of CCL18 release. Indeed, P2X7 was downregulated during spontaneous M2 differentiation, which may partially explain the ineffectiveness of late ATP stimulation of monocytes. However, pre-incubation of monocytes with PPADS, Suramin (unselective P2X- and P2Y-receptor blockers) and KN62 (P2X7-antagonist) failed to reverse the reduction of CCL18 by ATP. CONCLUSIONS: ATP prevents spontaneous differentiation of monocytes into M2-like macrophages in a dose- and time-dependent manner. These effects were not mediated by P2X and P2Y receptors.
Assuntos
Monócitos , Receptores Purinérgicos P2X7 , Humanos , Macrófagos , Diferenciação Celular , Trifosfato de Adenosina , Inflamação , Células CultivadasRESUMO
Sarcoidosis and chronic beryllium disease (CBD) are phenocopies, however the latter one has a clear trigger factor that is beryllium exposure. This study analyses single nucleotide polymorphisms (SNPs) in a large cohort for beryllium-exposed persons. SNPs were chosen for their relevance in sarcoidosis. Even though one of largest cohorts of beryllium-exposed persons was analysed, no statistically relevant association between any SNP and CBD could be verified. Notably, some SNPs exhibit inverse OR for beryllium sensitization and CBD with nominally statistical significance, which allows hypothesizing about pathophysiological role of genes for the disease triggering and development.
Assuntos
Beriliose/genética , Berílio/efeitos adversos , Butirofilinas/genética , DNA/genética , Exposição Ocupacional/efeitos adversos , Polimorfismo de Nucleotídeo Único , Beriliose/metabolismo , Butirofilinas/metabolismo , Doença Crônica , Feminino , Humanos , MasculinoAssuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos Imunológicos/efeitos adversos , Fentolamina/administração & dosagem , Pneumonia/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/administração & dosagem , Administração por Inalação , Idoso , Combinação de Medicamentos , Humanos , Imunoterapia/efeitos adversos , Pulmão/diagnóstico por imagem , Masculino , Melanoma/tratamento farmacológico , Pneumonia/induzido quimicamente , Pneumonia/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: The origin of collagen-producing cells in lung fibrosis is unclear. The involvement of embryonic signaling pathways has been acknowledged and trans-differentiation of epithelial cells is discussed critically. The work presented here investigates the role of TGFB in cytoskeleton remodeling and the expression of Epithelial-Mesenchymal-Transition markers by Alveolar Epithelial Cells Type II and tests the hypothesis if human alveolar epithelial cells are capable of trans-differentiation and production of pro-fibrotic collagen. METHODS: Primary human alveolar epithelial cells type II were extracted from donor tissues and stimulated with TGFß and a TGFß-inhibitor. Transcriptome and pathway analyses as well as validation of results on protein level were conducted. RESULTS: A TGFß-responsive fingerprint was found and investigated for mutual interactions. Interaction modules exhibited enrichment of genes that favor actin cytoskeleton remodeling, differentiation processes and collagen metabolism. Cross-validation of the TGFß-responsive fingerprint in an independent IPF dataset revealed overlap of genes and supported the direction of regulated genes and TGFß-specificity. CONCLUSIONS: Primary human alveolar epithelial cells type II seem undergo a TGFß-dependent phenotypic change, exhibit differential expression of EMT markers in vitro and acquire the potential to produce collagen.
Assuntos
Células Epiteliais Alveolares/metabolismo , Colágeno/biossíntese , Transição Epitelial-Mesenquimal/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Cultivadas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Sarcoidosis is a granulomatous disease that mainly affects the lung. A role of microbial factors in disease pathogenesis is assumed, but has not been investigated systematically in a large cohort.This cross-sectional study compared the lung microbiota of 71 patients with sarcoidosis, 15 patients with idiopathic pulmonary fibrosis (non-infectious controls) and 10 healthy controls (HCs). Next-generation sequencing of 16S DNA was used on bronchoalveolar lavage samples to characterise the microbial composition, which was analysed for diversity and indicator species. Host genotypes for 13 known sarcoidosis risk variants were determined and correlated with microbial parameters.The microbial composition differed significantly between sarcoidosis and HC samples (redundancy analysis ANOVA, p=0.025) and between radiographic Scadding types. Atopobium spp. was detected in 68% of sarcoidosis samples, but not in HC samples. Fusobacterium spp. was significantly more abundant in sarcoidosis samples compared with those from HCs. Mycobacteria were found in two of 71 sarcoidosis samples. Host-genotype analysis revealed an association of the rs2076530 (BTNL2) risk allele with a decrease in bacterial burden (p=0.002).Our results indicate Scadding type-dependent microbiota in sarcoidosis BAL samples. Atopobium spp. and Fusobacterium spp. were identified as sarcoidosis-associated bacteria, which may enable new insights into the pathogenesis and treatment of the disease.
Assuntos
Actinobacteria/isolamento & purificação , Fusobacterium/isolamento & purificação , Pulmão/microbiologia , Microbiota , Sarcoidose/microbiologia , Actinobacteria/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Líquido da Lavagem Broncoalveolar/microbiologia , Butirofilinas/genética , Estudos de Casos e Controles , Estudos Transversais , Feminino , Fusobacterium/genética , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Sarcoidose/genética , Adulto JovemRESUMO
Sarcoidosis is a granulomatous disease characterized by a T-helper type 1 (Th1) cell-dominated alveolitis. As a role of bacteria in the pathogenesis of sarcoidosis has been discussed, Toll-like receptors (TLRs) may be involved in the initiation of a first immune reaction. We analyzed expression and functional relevance of several TLRs in bronchoalveolar lavage (BAL) cells from patients with pulmonary sarcoidosis. In parallel, we determined the release of C-X-C motif chemokine 9 (CXCL9), CXCL10, and CXCL11 by BAL cells from patients with pulmonary sarcoidosis. Nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2, TLR2, TLR6, and TLR9 expression by BAL cells was analyzed by real-time RT-PCR and cell surface expression by flow cytometry. Chemokine release was measured in BAL cell culture supernatants by ELISA. We found increased TLR9 mRNA expression in patients with sarcoidosis with chest X-ray type I and II and TLR9 protein expression in BAL cells from patients with chest X-ray type II and III. Stimulation with CpG nucleotides increased CXCL10 release by BAL cells from patients with sarcoidosis type II significantly compared with control subjects or other patients with sarcoidosis. In contrast, no increase in TNF, IL-12p40, or CXCL8 was detected. Spontaneous release of CXCL10, but not CXCL9 or CXCL11, by cultured BAL cells was also highest in cells from patients with chest X-ray type II. We found a significant association between TLR9 expression and CD4+ lymphocytes in BAL. Our data demonstrate that TLR9 ligands may contribute to the immunopathogenesis of sarcoidosis via induction of CXCL10 release in the alveolar macrophages.
Assuntos
Quimiocina CXCL10/metabolismo , Receptores CXCR3/metabolismo , Sarcoidose Pulmonar/metabolismo , Receptor Toll-Like 9/metabolismo , Biópsia , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Ligantes , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Sarcoidose Pulmonar/patologiaRESUMO
RATIONALE: Pulmonary fibrosis is a progressive disease with only few treatment options available at the moment. Recently, the nucleoside uridine has been shown to exert anti-inflammatory effects in different animal models, e.g. in acute lung injury or bronchial asthma. METHOD: Therefore, we investigated the influence of uridine supplementation on inflammation and fibrosis in the classical bleomycin model. Male C57BL/6 mice received an intratracheal injection of bleomycin on day 0 and were treated intraperitoneally with uridine or vehicle. The degree of inflammation and fibrosis was assessed at different time points. RESULTS: Uridine administration resulted in attenuated inflammation, as demonstrated by reduced leukocytes and pro-inflammatory cytokines in the broncho-alveolar lavage (BAL) fluid. Furthermore, collagen deposition in the lung interstitium was also reduced by uridine supplementation. Similar results were obtained in a model in which animals received repeated intraperitoneal bleomycin injections. In addition uridine inhibited collagen and TGF-ß synthesis by primary lung fibroblasts, the release of pro-inflammatory cytokines by human lung epithelial cells, as well as the production of reactive oxygen species by human neutrophils. CONCLUSION: In summary, we were able to show that uridine has potent anti-inflammatory and anti-fibrotic properties. As uridine supplementation has been shown to be well tolerated and safe in humans, this might be a new therapeutic approach for the treatment of fibrotic lung diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Pulmão/efeitos dos fármacos , Pneumonia/prevenção & controle , Fibrose Pulmonar/prevenção & controle , Uridina/farmacologia , Animais , Bleomicina , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular Tumoral , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cystic fibrosis (CF) lung disease is characterised by chronic Pseudomonas aeruginosa infection and leukocyte infiltration. Chemokines recruit leukocytes to sites of infection. Gene expression analysis identified the chemokine CCL18 as upregulated in CF leukocytes. We hypothesised that CCL18 characterises infection and inflammation in patients with CF lung disease. Therefore, we quantified CCL18 protein levels in the serum and airway fluids of CF patients and healthy controls, and studied CCL18 protein production by airway cells ex vivo. These studies demonstrated that CCL18 levels were increased in the serum and airway fluids from CF patients compared with healthy controls. Within CF patients, CCL18 levels were increased in P. aeruginosa-infected CF patients. CCL18 levels in the airways, but not in serum, correlated with severity of pulmonary obstruction in CF. Airway cells isolated from P. aeruginosa-infected CF patients produced significantly higher amounts of CCL18 protein compared with airway cells from CF patients without P. aeruginosa infection or healthy controls. Collectively, these studies show that CCL18 levels characterise chronic P. aeruginosa infection and pulmonary obstruction in patients with CF. CCL18 may, thus, serve as a potential biomarker and therapeutic target in CF lung disease.
Assuntos
Quimiocinas CC/metabolismo , Fibrose Cística/metabolismo , Leucócitos/metabolismo , Infecções por Pseudomonas/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Quimiocina CXCL1/imunologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/imunologia , Quimiocina CXCL2/metabolismo , Quimiocinas CC/imunologia , Criança , Fibrose Cística/complicações , Fibrose Cística/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-8/imunologia , Interleucina-8/metabolismo , Leucócitos/imunologia , Masculino , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/imunologia , Escarro/metabolismo , Adulto JovemRESUMO
Sarcoidosis is a chronic granulomatous disorder characterized by an accumulation of lymphocytes and macrophages in the alveoli. Ultimately, long-lasting, nontreated disease results in a distortion of the microarchitecture of the lower respiratory tract. Our current understanding of its pathogenesis is that several sequential immunological events finally resulting in granuloma formation are involved: (1) dependent on a susceptible genetic background described by a variety of functional polymorphisms (2) the exposure to one or several still elusive antigen(s), leads to (3) an activation of macrophages, (4) an attainment of T cell immunity against the antigen(s) mediated by antigen processing and presentation by macrophages, and finally to (5) induction of granuloma formation. In this article, a detailed review on cellular and molecular mechanisms underpinning the sarcoid granulomatous lesion will be given. The important role of alveolar macrophages, T lymphocytes, regulatory T cells, and various cytokines/chemokines in orchestrating the induction, evolution, and immunoregulation of the sarcoid granulomatous/fibrotic lesions will be underscored. Although an etiological agent for sarcoidosis has not been identified, plausible "sarcoid antigens" including mycobacterial antigens such as mKatG or ESAT-6, antigens from Propionibacterium acnes, or even self-antigens will be discussed. It is possible that not one single causative agent exists but several germs, microbial products, or inorganic substances might induce pathogenetic mechanisms leading to a disease called sarcoidosis.
Assuntos
Linfócitos/imunologia , Macrófagos Alveolares/imunologia , Sarcoidose/imunologia , Animais , Antígenos/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Granuloma/imunologia , Granuloma/patologia , Humanos , Sarcoidose/fisiopatologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. METHODS: We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. RESULTS: We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. CONCLUSION: Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.
Assuntos
Fibrose Pulmonar Idiopática , Pulmão , Humanos , Camundongos , Animais , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibroblastos/metabolismo , Linhagem Celular , Colágeno/metabolismo , Quimiocinas CC/metabolismo , Receptores CCR6/metabolismoRESUMO
Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.
Assuntos
Mycobacterium tuberculosis , Pneumonia , Sarcoidose , Tuberculose , Humanos , Camundongos , Animais , Ligantes , Tuberculina , Ativinas , Imunoglobulina G , BiomarcadoresRESUMO
INTRODUCTION: While TLR ligands derived from microbial flora and pathogens are important activators of the innate immune system, a variety of factors such as intracellular bacteria, viruses, and parasites can induce a state of hyperreactivity, causing a dysregulated and potentially life-threatening cytokine over-response upon TLR ligand exposure. Type I interferon (IFN-αß) is a central mediator in the induction of hypersensitivity and is strongly expressed in splenic conventional dendritic cells (cDC) and marginal zone macrophages (MZM) when mice are infected with adenovirus. This study investigates the ability of adenoviral infection to influence the activation state of the immune system and underlines the importance of considering this state when planning the treatment of patients. METHODS: Infection with adenovirus-based vectors (Ad) or pretreatment with recombinant IFN-ß was used as a model to study hypersensitivity to lipopolysaccharide (LPS) in mice, murine macrophages, and human blood samples. The TNF-α, IL-6, IFN-αß, and IL-10 responses induced by LPS after pretreatment were measured. Mouse knockout models for MARCO, IFN-αßR, CD14, IRF3, and IRF7 were used to probe the mechanisms of the hypersensitive reaction. RESULTS: We show that, similar to TNF-α and IL-6 but not IL-10, the induction of IFN-αß by LPS increases strongly after Ad infection. This is true both in mice and in human blood samples ex vivo, suggesting that the regulatory mechanisms seen in the mouse are also present in humans. In mice, the scavenger receptor MARCO on IFN-αß-producing cDC and splenic marginal zone macrophages is important for Ad uptake and subsequent cytokine overproduction by LPS. Interestingly, not all IFN-αß-pretreated macrophage types exposed to LPS exhibit an enhanced TNF-α and IL-6 response. Pretreated alveolar macrophages and alveolar macrophage-like murine cell lines (MPI cells) show enhanced responses, while bone marrow-derived and peritoneal macrophages show a weaker response. This correlates with the respective absence or presence of the anti-inflammatory IL-10 response in these different macrophage types. In contrast, Ad or IFN-ß pretreatment enhances the subsequent induction of IFN-αß in all macrophage types. IRF3 is dispensable for the LPS-induced IFN-αß overproduction in infected MPI cells and partly dispensable in infected mice, while IRF7 is required. The expression of the LPS co-receptor CD14 is important but not absolutely required for the elicitation of a TNF-α over-response to LPS in Ad-infected mice. CONCLUSION: Viral infections or application of virus-based vaccines induces type I interferon and can tip the balance of the innate immune system in the direction of hyperreactivity to a subsequent exposure to TLR ligands. The adenoviral model presented here is one example of how multiple factors, both environmental and genetic, affect the physiological responses to pathogens. Being able to measure the current reactivity state of the immune system would have important benefits for infection-specific therapies and for the prevention of vaccination-elicited adverse effects.
Assuntos
Adenoviridae , Citocinas , Fator Regulador 3 de Interferon , Lipopolissacarídeos , Macrófagos , Camundongos Knockout , Animais , Camundongos , Lipopolissacarídeos/imunologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Macrófagos/imunologia , Citocinas/metabolismo , Camundongos Endogâmicos C57BL , Fator Regulador 7 de Interferon/metabolismo , Fator Regulador 7 de Interferon/genética , Vetores Genéticos , Infecções por Adenoviridae/imunologia , Interferon Tipo I/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Interferon beta/metabolismoRESUMO
Sarcoidosis is a systemic inflammatory disease of unknown aetiology, influenced by genetic and environmental factors. However, the loci so far identified for sarcoidosis explain only a part of its assumed heritability. To identify further susceptibility loci, we performed a genome-wide association analysis using the Affymetrix 6.0 Human GeneChip followed by validation and replication stages. After quality control, 637 cases, 1233 controls and 677 619 single-nucleotide polymorphisms (SNPs) were available for an initial screening. 99 SNPs were selected for validation in an independent study panel (1664 patients, 2932 controls). SNP rs1050045 was significantly associated with sarcoidosis (corrected p=0.0215) in the validation panel and yielded a p-value of 9.22 × 10(-8) (OR 1.24) in the meta-analysis of the screening and validation stage. A meta-analysis of three populations from Germany, the Czech Republic and Sweden confirmed this finding (p = 0.024; OR 1.14). Fine-mapping and mRNA expression studies pointed to osteosarcoma amplified 9 (OS9) as the most likely candidate for the underlying risk factor. The OS9 protein plays an important role in endoplasmic reticulum-associated protein degradation and acts during Toll-like receptor induced activation of myeloid cells. Expression analyses of OS9 mRNA provide evidence for a functional mechanism underlying the detected association signal.
Assuntos
Cromossomos Humanos Par 12 , Estudo de Associação Genômica Ampla , Pneumopatias/genética , Sarcoidose/genética , Estudos de Casos e Controles , Mapeamento Cromossômico/métodos , Doença Crônica , Predisposição Genética para Doença , Genótipo , Humanos , Pneumopatias/diagnóstico , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Fatores de Risco , Sarcoidose/diagnóstico , Análise de Sequência de DNARESUMO
RATIONALE: Sarcoidosis is a complex inflammatory disease with a heterogeneous clinical picture. Among others, an acute and chronic clinical course can be distinguished, for which specific genetic risk factors are known. OBJECTIVES: To identify additional risk loci for sarcoidosis and its acute and chronic subforms, we analyzed imputed data from a genome-wide association scan for these phenotypes. METHODS: After quality control, the genome-wide association scan comprised nearly 1.3 million imputed single-nucleotide polymorphisms based on an Affymetrix 6.0 Gene Chip dataset of 564 German sarcoidosis cases, including 176 acute and 354 chronic cases and 1,575 control subjects. MEASUREMENTS AND MAIN RESULTS: We identified chromosome 11q13.1 (rs479777) as a novel locus influencing susceptibility to sarcoidosis with genome-wide significance. The marker was significantly associated in three distinct German case-control populations and in an additional German family sample with odds ratios ranging from 0.67 to 0.77. This finding was further replicated in two independent European case-control populations from the Czech Republic (odds ratio, 0.75) and from Sweden (odds ratio, 0.79). In a meta-analysis of the included European case-control samples the marker yielded a P value of 2.68 × 10(-18). The locus was previously reported to be associated with Crohn disease, psoriasis, alopecia areata, and leprosy. For sarcoidosis, fine-mapping and expression analysis suggest KCNK4, PRDX5, PCLB3, and most promising CCDC88B as candidates for the underlying risk gene in the associated region. CONCLUSIONS: This study provides striking evidence for association of chromosome 11q13.1 with sarcoidosis in Europeans, and thus identified a further genetic risk locus shared by sarcoidosis, Crohn disease and psoriasis.
Assuntos
Proteínas de Transporte/genética , Doença de Crohn/genética , Sarcoidose/genética , Doença Aguda , Estudos de Casos e Controles , Mapeamento Cromossômico , Doença Crônica , República Tcheca , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Alemanha , Humanos , Polimorfismo de Nucleotídeo Único , SuéciaRESUMO
Introduction: Sarcoidosis is a highly variable disease in terms of organ involvement, type of onset and course. Associations of genetic polymorphisms with sarcoidosis phenotypes have been observed and suggest genetic signatures. Methods: After obtaining a positive vote of the competent ethics committee we genotyped 1909 patients of the deeply phenotyped Genetic-Phenotype Relationship in Sarcoidosis (GenPhenReSa) cohort of 31 European centers in 12 countries with 116 potentially disease-relevant single-nucleotide polymorphisms (SNPs). Using a meta-analysis, we investigated the association of relevant phenotypes (acute vs. sub-acute onset, phenotypes of organ involvement, specific organ involvements, and specific symptoms) with genetic markers. Subgroups were built on the basis of geographical, clinical and hospital provision considerations. Results: In the meta-analysis of the full cohort, there was no significant genetic association with any considered phenotype after correcting for multiple testing. In the largest sub-cohort (Serbia), we confirmed the known association of acute onset with TNF and reported a new association of acute onset an HLA polymorphism. Multi-locus models with sets of three SNPs in different genes showed strong associations with the acute onset phenotype in Serbia and Lublin (Poland) demonstrating potential region-specific genetic links with clinical features, including recently described phenotypes of organ involvement. Discussion: The observed associations between genetic variants and sarcoidosis phenotypes in subgroups suggest that gene-environment-interactions may influence the clinical phenotype. In addition, we show that two different sets of genetic variants are permissive for the same phenotype of acute disease only in two geographic subcohorts pointing to interactions of genetic signatures with different local environmental factors. Our results represent an important step towards understanding the genetic architecture of sarcoidosis.
RESUMO
Extracellular ATP acts as a "danger signal" and can induce inflammation by binding to purinergic receptors. Chronic obstructive pulmonary disease is one of the most common inflammatory diseases associated with cigarette smoke inhalation, but the underlying mechanisms are incompletely understood. In this study, we show that endogenous pulmonary ATP levels are increased in a mouse model of smoke-induced acute lung inflammation and emphysema. ATP neutralization or nonspecific P2R-blockade markedly reduced smoke-induced lung inflammation and emphysema. We detected an upregulation the purinergic receptors subtypes on neutrophils (e.g., P2Y2R), macrophages, and lung tissue from animals with smoke-induced lung inflammation. By using P2Y(2)R deficient ((-/-)) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y(2)R. Moreover, P2Y(2)R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y(2)R(-/-) and wild type chimera animals revealed that P2Y(2)R expression on hematopoietic cell plays the pivotal role in the observed effect. We demonstrate, for the first time, that endogenous ATP contributes to smoke-induced lung inflammation and then development of emphysema via activation of the purinergic receptor subtypes, such as P2Y(2)R.
Assuntos
Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/prevenção & controle , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiologia , Lesão por Inalação de Fumaça/metabolismo , Lesão por Inalação de Fumaça/prevenção & controle , Doença Aguda , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/fisiologia , Animais , Movimento Celular/imunologia , Doença Crônica , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Enfisema Pulmonar/patologia , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2Y2 , Lesão por Inalação de Fumaça/patologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
RATIONALE: Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated. OBJECTIVES: To investigate the role of P2Y6R in asthma pathogenesis. METHODS: Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system. MEASUREMENTS AND MAIN RESULTS: We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro. CONCLUSIONS: P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.
Assuntos
Remodelação das Vias Aéreas/imunologia , Inflamação/imunologia , Pulmão/imunologia , Receptores Purinérgicos/imunologia , Hipersensibilidade Respiratória/imunologia , Compostos de Alúmen , Análise de Variância , Animais , Células Cultivadas , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , OvalbuminaRESUMO
Extracellular ATP is up-regulated in the airways of patients with chronic obstructive pulmonary disease, and may contribute to the pathogenesis of the disease. However, the precise mechanisms are poorly understood. Our objective was to investigate the functional role of the ATP receptor P2X(7) in the pathogenesis of cigarette smoke (CS)-induced lung inflammation and emphysema in vivo. Expression of the P2X(7) receptor (P2X(7)R) was measured in lung tissue und immune cells of mice with CS-induced lung inflammation. In a series of experiments using P2X(7) antagonists and genetically engineered mice, the functional role of the P2X(7)R in CS-induced lung inflammation was explored. CS-induced inflammation was associated with an up-regulation of the P2X(7)R on blood and airway neutrophils, alveolar macrophages, and in whole lung tissue. Selective intrapulmonary inhibition of the P2X(7)R reduced CS-induced lung inflammation and prevented the development of emphysema. Accordingly, P2X(7)R knockout mice showed a reduced pulmonary inflammation after acute CS exposure. Experiments with P2X(7)R chimera animals revealed that immune cell P2X(7)R expression plays an important role in CS-induced lung inflammation and emphysema. Extracellular ATP contributes to the development of CS-induced lung inflammation and emphysema via activation of the P2X(7)R. Inhibition of this receptor may be a new therapeutic target for the treatment of chronic obstructive pulmonary disease.