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1.
Immunity ; 54(12): 2908-2921.e6, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34788600

RESUMO

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.


Assuntos
Betacoronavirus/fisiologia , Vacinas contra COVID-19/imunologia , Infecções por Coronavirus/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Sequência Conservada/genética , Evolução Molecular , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Desenvolvimento de Vacinas
2.
Am J Respir Crit Care Med ; 207(5): 553-565, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36170617

RESUMO

Rationale: Tissue-resident natural killer (trNK) cells have been identified in numerous organs, but little is known about their functional contribution to respiratory immunity, in particular during chronic lung diseases such as chronic obstructive pulmonary disease (COPD). Objectives: To investigate the phenotype and antiviral responses of trNK cells in murine cigarette smoke-induced experimental COPD and in human lung parenchyma from COPD donors. Methods: Mice were exposed to cigarette smoke for 12 weeks to induce COPD-like lung disease. Lung trNK cell phenotypes and function were analyzed by flow cytometry in both murine and human disease with and without challenge with influenza A virus. Measurements and Main Results: In the mouse lung, CD49a+CD49b+EOMES+ and CD49a+CD49b-EOMESlo NK cell populations had a distinct phenotype compared with CD49a- circulating NK cells. CD49a+ NK cells were more extensively altered earlier in disease onset than circulating NK cells, and increased proportions of CD49a+ NK cells correlated with worsening disease in both murine and human COPD. Furthermore, the presence of lung disease delayed both circulating and trNK cell functional responses to influenza infection. CD49a+ NK cells markedly increased their NKG2D, CD103, and CD69 expression in experimental COPD after influenza infection, and human CD49a+ NK cells were hyperactive to ex vivo influenza infection in COPD donors. Conclusions: Collectively, these results demonstrate that trNK cell function is altered in cigarette smoke-induced disease and suggests that smoke exposure may aberrantly prime trNK cell responsiveness to viral infection. This may contribute to excess inflammation during viral exacerbations of COPD.


Assuntos
Influenza Humana , Pneumopatias , Doença Pulmonar Obstrutiva Crônica , Humanos , Camundongos , Animais , Integrina alfa1/metabolismo , Influenza Humana/metabolismo , Integrina alfa2/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Células Matadoras Naturais , Pulmão/metabolismo , Pneumopatias/metabolismo , Antivirais
3.
Eur Respir J ; 61(4)2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36549711

RESUMO

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.


Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Camundongos , Animais , Pulmão , Morte Celular , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
4.
Respir Res ; 24(1): 303, 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38044426

RESUMO

BACKGROUND: Increased airway NLRP3 inflammasome-mediated IL-1ß responses may underpin severe neutrophilic asthma. However, whether increased inflammasome activation is unique to severe asthma, is a common feature of immune cells in all inflammatory types of severe asthma, and whether inflammasome activation can be therapeutically targeted in patients, remains unknown. OBJECTIVE: To investigate the activation and inhibition of inflammasome-mediated IL-1ß responses in immune cells from patients with asthma. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with non-severe (n = 59) and severe (n = 36 stable, n = 17 exacerbating) asthma and healthy subjects (n = 39). PBMCs were stimulated with nigericin or lipopolysaccharide (LPS) alone, or in combination (LPS + nigericin), with or without the NLRP3 inhibitor MCC950, and the effects on IL-1ß release were assessed. RESULTS: PBMCs from patients with non-severe or severe asthma produced more IL-1ß in response to nigericin than those from healthy subjects. PBMCs from patients with severe asthma released more IL-1ß in response to LPS + nigericin than those from non-severe asthma. Inflammasome-induced IL-1ß release from PBMCs from patients with severe asthma was not increased during exacerbation compared to when stable. Inflammasome-induced IL-1ß release was not different between male and female, or obese and non-obese patients and correlated with eosinophil and neutrophil numbers in the airways. MCC950 effectively suppressed LPS-, nigericin-, and LPS + nigericin-induced IL-1ß release from PBMCs from all groups. CONCLUSION: An increased ability for inflammasome priming and/or activation is a common feature of systemic immune cells in both severe and non-severe asthma, highlighting inflammasome inhibition as a universal therapy for different subtypes of disease.


Assuntos
Asma , Inflamassomos , Humanos , Masculino , Feminino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nigericina/farmacologia , Lipopolissacarídeos , Leucócitos Mononucleares , Interleucina-1beta , Asma/diagnóstico , Asma/tratamento farmacológico , Sulfonamidas
5.
Am J Respir Crit Care Med ; 206(6): 712-729, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35549656

RESUMO

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.


Assuntos
COVID-19 , Doença Pulmonar Obstrutiva Crônica , Serpinas , Citocinas , Células Epiteliais , Humanos , Peptídeo Hidrolases , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , SARS-CoV-2 , Análise de Sequência de RNA , Serpinas/farmacologia , Serpinas/uso terapêutico
6.
Int J Mol Sci ; 24(4)2023 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-36834913

RESUMO

Lung cancer is the leading cause of cancer-related deaths globally, in part due to a lack of early diagnostic tools and effective pharmacological interventions. Extracellular vesicles (EVs) are lipid-based membrane-bound particles released from all living cells in both physiological and pathological states. To understand the effects of lung-cancer-derived EVs on healthy cells, we isolated and characterized EVs derived from A549 lung adenocarcinoma cells and transferred them to healthy human bronchial epithelial cells (16HBe14o). We found that A549-derived EVs carry oncogenic proteins involved in the pathway of epithelial to mesenchymal transition (EMT) that are regulated by ß-catenin. The exposure of 16HBe14o cells to A549-derived EVs resulted in a significant increase in cell proliferation, migration, and invasion via upregulating EMT markers such as E-Cadherin, Snail, and Vimentin and cell adhesion molecules such as CEACAM-5, ICAM-1, and VCAM-1, with concomitant downregulation of EpCAM. Our study suggests a role for cancer-cell-derived EVs to induce tumorigenesis in adjacent healthy cells by promoting EMT via ß-catenin signaling.


Assuntos
Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , beta Catenina/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal , Vesículas Extracelulares/metabolismo , Neoplasias Pulmonares/metabolismo , Transdução de Sinais
7.
Eur Respir J ; 60(6)2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35777766

RESUMO

BACKGROUND: COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. METHODS: We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. RESULTS: The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CONCLUSION: CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.


Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Animais , Camundongos , Quimases/metabolismo , Mastócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Remodelação das Vias Aéreas , Enfisema Pulmonar/etiologia , Pulmão , Enfisema/complicações , Inflamação/metabolismo , Camundongos Endogâmicos C57BL
8.
Int J Mol Sci ; 24(1)2022 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-36613693

RESUMO

Bifidobacterium are prominent gut commensals that produce the short-chain fatty acid (SCFA) acetate, and they are often used as probiotics. Connections between the gut and the lung, termed the gut-lung axis, are regulated by the microbiome. The gut-lung axis is increasingly implicated in cigarette smoke-induced diseases, and cigarette smoke exposure has been associated with depletion of Bifidobacterium species. In this study, we assessed the impact of acetate-producing Bifidobacterium longum subsp. longum (WT) and a mutant strain with an impaired acetate production capacity (MUT) on cigarette smoke-induced inflammation. The mice were treated with WT or MUT B. longum subsp. longum and exposed to cigarette smoke for 8 weeks before assessments of lung inflammation, lung tissue gene expression and cecal SCFAs were performed. Both strains of B. longum subsp. longum reduced lung inflammation, inflammatory cytokine expression and adhesion factor expression and alleviated cigarette smoke-induced depletion in caecum butyrate. Thus, the probiotic administration of B. longum subsp. longum, irrespective of its acetate-producing capacity, alleviated cigarette smoke-induced inflammation and the depletion of cecal butyrate levels.


Assuntos
Fumar Cigarros , Probióticos , Camundongos , Animais , Bifidobacterium , Probióticos/farmacologia , Butiratos , Acetatos , Inflamação
9.
Clin Exp Allergy ; 51(1): 120-131, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33098152

RESUMO

BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.


Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Asma/metabolismo , Interleucina-13/metabolismo , Interleucina-9/metabolismo , Complacência Pulmonar/efeitos dos fármacos , Pulmão/efeitos dos fármacos , beta-Defensinas/farmacologia , Animais , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/fisiopatologia , Chlamydia muridarum , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/fisiopatologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos , Ovalbumina , Pyroglyphidae , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/fisiopatologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/fisiopatologia
10.
Inflammopharmacology ; 29(1): 15-33, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33152094

RESUMO

Asthma is a common, heterogeneous and serious disease, its prevalence has steadily risen in most parts of the world, and the condition is often inadequately controlled in many patients. Hence, there is a major need for new therapeutic approaches. Mild-to-moderate asthma is considered a T-helper cell type-2-mediated inflammatory disorder that develops due to abnormal immune responses to otherwise innocuous allergens. Prolonged exposure to allergens and persistent inflammation results in myofibroblast infiltration and airway remodelling with mucus hypersecretion, airway smooth muscle hypertrophy, and excess collagen deposition. The airways become hyper-responsive to provocation resulting in the characteristic wheezing and obstructed airflow experienced by patients. Extensive research has progressed the understanding of the underlying mechanisms and the development of new treatments for the management of asthma. Here, we review the basis of the disease, covering new areas such as the role of vascularisation and microRNAs, as well as associated potential therapeutic interventions utilising reports from animal and human studies. We also cover novel drug delivery strategies that are being developed to enhance therapeutic efficacy and patient compliance. Potential avenues to explore to improve the future of asthma management are highlighted.


Assuntos
Alérgenos/imunologia , Antiasmáticos/administração & dosagem , Asma/tratamento farmacológico , Remodelação das Vias Aéreas , Animais , Asma/genética , Asma/imunologia , Sistemas de Liberação de Medicamentos , Humanos , Adesão à Medicação , MicroRNAs/genética
11.
Immunol Rev ; 278(1): 41-62, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28658552

RESUMO

Severe, steroid-resistant asthma is clinically and economically important since affected individuals do not respond to mainstay corticosteroid treatments for asthma. Patients with this disease experience more frequent exacerbations of asthma, are more likely to be hospitalized, and have a poorer quality of life. Effective therapies are urgently required, however, their development has been hampered by a lack of understanding of the pathological processes that underpin disease. A major obstacle to understanding the processes that drive severe, steroid-resistant asthma is that the several endotypes of the disease have been described that are characterized by different inflammatory and immunological phenotypes. This heterogeneity makes pinpointing processes that drive disease difficult in humans. Clinical studies strongly associate specific respiratory infections with severe, steroid-resistant asthma. In this review, we discuss key findings from our studies where we describe the development of representative experimental models to improve our understanding of the links between infection and severe, steroid-resistant forms of this disease. We also discuss their use in elucidating the mechanisms, and their potential for developing effective therapeutic strategies, for severe, steroid-resistant asthma. Finally, we highlight how the immune mechanisms and therapeutic targets we have identified may be applicable to obesity-or pollution-associated asthma.


Assuntos
Antiasmáticos/uso terapêutico , Asma/diagnóstico , Asma/terapia , Resistência a Medicamentos , Hipersensibilidade Respiratória/diagnóstico , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/terapia , Esteroides/uso terapêutico , Poluição do Ar , Animais , Asma/etiologia , Asma/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Obesidade/complicações , Fatores de Risco , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
12.
Clin Exp Allergy ; 50(1): 82-94, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31579973

RESUMO

BACKGROUND: Acute exacerbations of asthma represent a major burden of disease and are often caused by respiratory infections. Viral infections are recognized as significant triggers of exacerbations; however, less is understood about the how microbial bioproducts such as the endotoxin (lipopolysaccharide (LPS)) trigger episodes. Indeed, increased levels of LPS have been linked to asthma onset, severity and steroid resistance. OBJECTIVE: The goal of this study was to identify mechanisms underlying bacterial-induced exacerbations by employing LPS as a surrogate for infection. METHODS: We developed a mouse model of LPS-induced exacerbation on the background of pre-existing type-2 allergic airway disease (AAD). RESULTS: LPS-induced exacerbation was characterized by steroid-resistant airway hyperresponsiveness (AHR) and an exaggerated inflammatory response distinguished by increased numbers of infiltrating neutrophils/macrophages and elevated production of lung inflammatory cytokines, including TNFα, IFNγ, IL-27 and MCP-1. Expression of the type-2 associated inflammatory factors such as IL-5 and IL-13 were elevated in AAD but not altered by LPS exposure. Furthermore, AHR and airway inflammation were no longer suppressed by corticosteroid (dexamethasone) treatment after LPS exposure. Depletion of pulmonary macrophages by administration of 2-chloroadenosine into the lungs suppressed AHR and reduced IL-13, TNFα and IFNγ expression. Blocking IL-13 function, through either IL-13-deficiency or administration of specific blocking antibodies, also suppressed AHR and airway inflammation. CONCLUSIONS & CLINICAL RELEVANCE: We present evidence that IL-13 and innate immune pathways (in particular pulmonary macrophages) contribute to LPS-induced exacerbation of pre-existing AAD and provide insight into the complex molecular processes potentially underlying microbial-induced exacerbations.


Assuntos
Asma/imunologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Interleucina-13/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Hipersensibilidade Respiratória/imunologia , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Infecções Bacterianas , Líquido da Lavagem Broncoalveolar/citologia , Quimiocina CCL2 , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Modelos Animais de Doenças , Progressão da Doença , Resistência a Medicamentos , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Interleucinas/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Mucina-5AC/efeitos dos fármacos , Mucina-5AC/metabolismo , Ovalbumina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia
13.
BMC Cancer ; 20(1): 217, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171282

RESUMO

BACKGROUND: Colorectal cancer (CRC) is the most common cancer and a leading cause of death worldwide. Extracellular matrix (ECM) proteins regulate tumor growth and development in CRC. Elastin (ELN) is a component of ECM proteins involved in the tumor microenvironment. However, the role of ELN in CRC remains unclear. METHODS: In this study, we analyzed ELN gene expression in tumors from CRC patients and adjacent non-tumor colon tissues and healthy controls from two existing microarray datasets. ELN protein was measured in human normal colon cells and colon cancer epithelial cells and tumor development was assessed in colon epithelial cells cultured in medium with or without ELN peptide on plates coated with ELN recombinant protein. Control plates were coated with PBS only. RESULTS: We found ELN gene expression was increased in tumors from CRC patients compared to adjacent non-tumor tissues and healthy controls. ELN protein was increased in cancer cells compared to normal colon epithelial cells. Transforming growth factor beta (TGF-ß) was a key cytokine to induce production of ECM proteins, but it did not induce ELN expression in colon cancer cells. Matrix metalloproteinase 9 (MMP9) gene expression was increased, but that of MMP12 (elastase) did not change between CRC patients and control. Tissue inhibitor of metalloproteinases 3 (TIMP3) gene expression was decreased in colon tissues from CRC patients compared to healthy controls. However, MMP9, MMP12 and TIMP3 proteins were increased in colon cancer cells. ELN recombinant protein increased proliferation and wound healing in colon cancer epithelial cells. This had further increased in cancer cells incubated in plates coated with recombinant ELN coated plate and in culture media containing ELN peptide. A potential mechanism was that ELN induced epithelial mesenchymal transition with increased alpha-smooth muscle actin and vimentin proteins but decreased E-cadherin protein. Tumor necrosis factor alpha (TNF) mRNA was also increased in CRC patients compared to controls. ELN recombinant protein induced further increases in TNF protein in mouse bone marrow derived macrophages after lipopolysaccharide stimulation. CONCLUSIONS: These data suggest ELN regulates tumor development and the microenvironment in CRC.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Elastina/genética , Elastina/metabolismo , Animais , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
Eur Respir J ; 54(1)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31196943

RESUMO

Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death globally. The lack of effective treatments results from an incomplete understanding of the underlying mechanisms driving COPD pathogenesis.Interleukin (IL)-22 has been implicated in airway inflammation and is increased in COPD patients. However, its roles in the pathogenesis of COPD is poorly understood. Here, we investigated the role of IL-22 in human COPD and in cigarette smoke (CS)-induced experimental COPD.IL-22 and IL-22 receptor mRNA expression and protein levels were increased in COPD patients compared to healthy smoking or non-smoking controls. IL-22 and IL-22 receptor levels were increased in the lungs of mice with experimental COPD compared to controls and the cellular source of IL-22 included CD4+ T-helper cells, γδ T-cells, natural killer T-cells and group 3 innate lymphoid cells. CS-induced pulmonary neutrophils were reduced in IL-22-deficient (Il22 -/-) mice. CS-induced airway remodelling and emphysema-like alveolar enlargement did not occur in Il22 -/- mice. Il22 -/- mice had improved lung function in terms of airway resistance, total lung capacity, inspiratory capacity, forced vital capacity and compliance.These data highlight important roles for IL-22 and its receptors in human COPD and CS-induced experimental COPD.


Assuntos
Enfisema/etiologia , Interleucinas/fisiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores de Interleucina/fisiologia , Remodelação das Vias Aéreas , Resistência das Vias Respiratórias , Animais , Enfisema/patologia , Feminino , Humanos , Imunidade Inata , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Produtos do Tabaco , Interleucina 22
15.
Crit Rev Eukaryot Gene Expr ; 28(2): 125-127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30055538

RESUMO

MicroRNAs (miRNAs) are short noncoding RNAs that control gene expression posttranscriptionally by directly blocking translation of their target mRNAs or by repressing protein production via mRNA destabilization. Investigations into miRNAs began approximately 12 years ago with their discovery in mammalian cells. Still, the involvement of miRNAs in the development of asthma remains unclear, and this topic needs further research to discover various molecular mechanisms responsible for the pathogenesis of asthma and new therapeutic interventions. So far, various miRNAs have been identified in allergic airway disease along with their targets. Our present mini-review highlights the latest information involving the role of miRNAs in asthma.


Assuntos
Asma/genética , MicroRNAs/genética , Hipersensibilidade Respiratória/genética , Asma/patologia , Regulação da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , Hipersensibilidade Respiratória/patologia , Sistema Respiratório/patologia
16.
J Pathol ; 243(4): 510-523, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28862768

RESUMO

Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin-1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)-induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c-deficient (Fbln1c-/- ) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild-type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c-/- mice with AAD also had reduced numbers of α-smooth muscle actin-positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)-5, IL-13, IL-33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL-5, IL-33 and TNF levels in lung-draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3-positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL-5 and IL-13 from co-cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium-treated controls. Our data show that Fbln1c may be a therapeutic target in chronic asthma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Hiper-Reatividade Brônquica/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Actinas/metabolismo , Animais , Asma/imunologia , Asma/fisiopatologia , Asma/prevenção & controle , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/prevenção & controle , Broncoconstrição , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Feminino , Genótipo , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/fisiopatologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Interferência de RNA , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Transfecção
17.
Am J Respir Crit Care Med ; 196(3): 283-297, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28252317

RESUMO

RATIONALE: Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1ß responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma. OBJECTIVES: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1ß in severe, steroid-resistant asthma. METHODS: We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1ß responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1ß responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1ß antibody. Roles for IL-1ß-induced neutrophilic inflammation were examined using IL-1ß and anti-Ly6G. MEASUREMENTS AND MAIN RESULTS: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1ß responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1ß expression. Treatment with anti-IL-1ß, Ac-YVAD-cho, and MCC950 suppressed IL-1ß responses and the important steroid-resistant features of disease in mice, whereas IL-1ß administration recapitulated these features. Neutrophil depletion suppressed IL-1ß-induced steroid-resistant airway hyperresponsiveness. CONCLUSIONS: NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.


Assuntos
Asma/genética , Asma/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Animais , Asma/metabolismo , Modelos Animais de Doenças , Resistência a Medicamentos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Índice de Gravidade de Doença , Resultado do Tratamento
18.
J Allergy Clin Immunol ; 139(2): 519-532, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27448447

RESUMO

BACKGROUND: Severe steroid-insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid-insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid-insensitive patients with eosinophil-enriched inflammation have also been described. The mechanisms that underpin infection-induced, severe steroid-insensitive asthma can be elucidated by using mouse models of disease. OBJECTIVE: We sought to develop representative mouse models of severe, steroid-insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. METHODS: Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin-induced, severe, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. RESULTS: Infection induced increases in the levels of microRNA (miRNA)-21 (miR-21) expression in the lung during SSIAAD, whereas expression of the miR-21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3-kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR-21-specific antagomir (Ant-21) increased phosphatase and tensin homolog levels. Treatment with Ant-21, or the pan-PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. CONCLUSION: We identify a previously unrecognized role for an miR-21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR-21 as a novel therapeutic target for the treatment of this form of asthma.


Assuntos
Asma/genética , Chlamydia muridarum/imunologia , Haemophilus influenzae/imunologia , Histona Desacetilase 2/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Pneumonia/genética , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/genética , Animais , Antagomirs/genética , Asma/tratamento farmacológico , Asma/imunologia , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Resistência a Medicamentos , Regulação da Expressão Gênica , Histona Desacetilase 2/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/imunologia
19.
J Immunol ; 193(8): 4072-82, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25200951

RESUMO

Respiratory virus infections are often pathogenic, driving severe inflammatory responses. Most research has focused on localized effects of virus infection and inflammation. However, infection can induce broad-reaching, systemic changes that are only beginning to be characterized. In this study, we assessed the impact of acute pneumovirus infection in C57BL/6 mice on bone marrow hematopoiesis. We hypothesized that inflammatory cytokine production in the lung upregulates myeloid cell production in response to infection. We demonstrate a dramatic increase in the percentages of circulating myeloid cells, which is associated with pronounced elevations in inflammatory cytokines in serum (IFN-γ, IL-6, CCL2), bone (TNF-α), and lung tissue (TNF-α, IFN-γ, IL-6, CCL2, CCL3, G-CSF, osteopontin). Increased hematopoietic stem/progenitor cell percentages (Lineage(-)Sca-I(+)c-kit(+)) were also detected in the bone marrow. This increase was accompanied by an increase in the proportions of committed myeloid progenitors, as determined by colony-forming unit assays. However, no functional changes in hematopoietic stem cells occurred, as assessed by competitive bone marrow reconstitution. Systemic administration of neutralizing Abs to either TNF-α or IFN-γ blocked expansion of myeloid progenitors in the bone marrow and also limited virus clearance from the lung. These findings suggest that acute inflammatory cytokines drive production and differentiation of myeloid cells in the bone marrow by inducing differentiation of committed myeloid progenitors. Our findings provide insight into the mechanisms via which innate immune responses regulate myeloid cell progenitor numbers in response to acute respiratory virus infection.


Assuntos
Diferenciação Celular/imunologia , Citocinas/imunologia , Células Progenitoras Mieloides/citologia , Infecções por Pneumovirus/imunologia , Pneumovirus , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Células da Medula Óssea/citologia , Proliferação de Células , Citocinas/sangue , Hematopoese , Imunidade Inata , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/imunologia
20.
J Allergy Clin Immunol ; 131(3): 752-62, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23380220

RESUMO

BACKGROUND: Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES: We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS: Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS: After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION: A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.


Assuntos
Modelos Animais de Doenças , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumaça/efeitos adversos , Triptases/imunologia , Remodelação das Vias Aéreas , Animais , Macrófagos/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Testes de Função Respiratória , Nicotiana , Triptases/deficiência , Triptases/genética
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