RESUMO
Acute respiratory distress syndrome (ARDS) remains a significant problem in need of new pharmaceutical approaches to improve its resolution. Studies comparing gene expression signatures in rodents and humans with lung injury reveal conserved pathways, including MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-related protein kinase) activation. In preclinical acute lung injury (ALI) models, inhibition of MAP2K1 (MAPK kinase 1)/MAP2K2 (MAPK kinase 2) improves measures of ALI. Myeloid cell deletion of MAP2K1 results in sustained MAP2K2 activation and nonresolving ALI, suggesting that MAP2K2 deactivation may be a key driver of ALI resolution. We used human genomic data from the iSPAAR (Identification of SNPs Predisposing to Altered Acute Lung Injury Risk) Consortium to assess genetic variants in MAP2K1 and MAP2K2 for association with mortality from ARDS. To determine the role of MAP2K2 in ALI recovery, we studied mice deficient in Map2k2 (Mek2-/-) and wild-type control mice in ALI models. We identified a MAP2K2 variant that was associated with death in ARDS and MAP2K2 expression. In Pseudomonas aeruginosa ALI, Mek2-/- mice had similar early alveolar neutrophilic recruitment but faster resolution of alveolar neutrophilia and vascular leak. Gene expression analysis revealed a role for MAP2K2 in promoting and sustaining select proinflammatory pathway activation in ALI. Bone marrow chimera studies indicate that leukocyte MAP2K2 is the key regulator of ALI duration. These studies implicate a role for MAP2K2 in ALI duration via transcriptional regulation of inflammatory programming with potential relevance to ARDS. Targeting leukocyte MAP2K2 may be an effective strategy to promote ALI resolution.
Assuntos
Lesão Pulmonar Aguda , MAP Quinase Quinase 2/metabolismo , Síndrome do Desconforto Respiratório , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , MAP Quinase Quinase 2/genética , Camundongos , Síndrome do Desconforto Respiratório/genéticaRESUMO
Coxiella burnetii, a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii-infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1ß secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1ß and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii, T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever.
Assuntos
Coxiella burnetii , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Febre Q , Animais , Camundongos , Camundongos Endogâmicos C57BL , Febre Q/imunologiaRESUMO
Syndecan-1 is a transmembrane proteoglycan expressed prominently by lung epithelium and has pleiotropic functions such as regulating cell migration, proliferation, and survival. Loss of syndecan-1 expression by lung cancer cells is associated with higher-grade cancers and worse clinical prognosis. We evaluated the effects of syndecan-1 in various cell-based and animal models of lung cancer and found that lung tumorigenesis was moderated by syndecan-1. We also demonstrate that syndecan-1 (or lack thereof) alters the miRNA cargo carried within exosomes exported from lung cancer cells. Analysis of the changes in miRNA expression identified a distinct shift toward augmented procancer signaling consistent with the changes found in lung adenocarcinoma. Collectively, our work identifies syndecan-1 as an important factor in lung cancer cells that shapes the tumor microenvironment through alterations in miRNA packaging within exosomes.
Assuntos
Carcinogênese/metabolismo , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , Sindecana-1/metabolismo , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Proliferação de Células , Regulação para Baixo/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/patologia , Camundongos , MicroRNAs/metabolismo , Análise de Sobrevida , Regulação para Cima/genéticaRESUMO
Several members of the matrix metalloproteinase (MMP) family control a range of immune processes, such as leukocyte influx and chemokine activity. Stromelysin-2 (MMP10) is expressed by macrophages in numerous tissues after injury; however, little is known of its function. In this study, we report that MMP10 is expressed by macrophages in human lungs from patients with cystic fibrosis and induced in mouse macrophages in response to Pseudomonas aeruginosa infection both in vivo and by isolated resident alveolar and bone marrow-derived macrophages (BMDM). Our data indicates that macrophage MMP10 serves a beneficial function in response to acute infection. Whereas wild-type mice survived infection with minimal morbidity, 50% of Mmp10(-/-) mice died and all showed sustained weight loss (morbidity). Although bacterial clearance and neutrophil influx did not differ between genotypes, macrophage numbers were â¼3-fold greater in infected Mmp10(-/-) lungs than in wild-types. Adoptive transfer of wild-type BMDM normalized infection-induced morbidity in Mmp10(-/-) recipients to wild-type levels, demonstrating that the protective effect of MMP10 was due to its production by macrophages. Both in vivo and in cultured alveolar macrophages and BMDM, expression of several M1 macrophage markers was elevated, whereas M2 markers were reduced in Mmp10(-/-) tissue and cells. Global gene expression analysis revealed that infection-mediated transcriptional changes persisted in Mmp10(-/-) BMDM long after they were downregulated in wild-type cells. These results indicate that MMP10 serves a beneficial role in response to acute infection by moderating the proinflammatory response of resident and infiltrating macrophages.
Assuntos
Fibrose Cística/imunologia , Inflamação/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Metaloproteinase 10 da Matriz/imunologia , Transferência Adotiva , Animais , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Pseudomonas/imunologiaRESUMO
Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.
Assuntos
Inflamação/microbiologia , Inflamação/virologia , Inovirus/crescimento & desenvolvimento , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/virologia , Pseudomonas aeruginosa/virologia , Animais , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/microbiologia , Fibrose Cística/virologia , Pulmão/microbiologia , Pulmão/virologia , Macrófagos/microbiologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologiaRESUMO
Chronic respiratory disorders are important contributors to the global burden of disease. Genome-wide association studies (GWASs) of lung function measures have identified several trait-associated loci, but explain only a modest portion of the phenotypic variability. We postulated that integrating pathway-based methods with GWASs of pulmonary function and airflow obstruction would identify a broader repertoire of genes and processes influencing these traits. We performed two independent GWASs of lung function and applied gene set enrichment analysis to one of the studies and validated the results using the second GWAS. We identified 131 significantly enriched gene sets associated with lung function and clustered them into larger biological modules involved in diverse processes including development, immunity, cell signaling, proliferation and arachidonic acid. We found that enrichment of gene sets was not driven by GWAS-significant variants or loci, but instead by those with less stringent association P-values. Next, we applied pathway enrichment analysis to a meta-analyzed GWAS of airflow obstruction. We identified several biologic modules that functionally overlapped with those associated with pulmonary function. However, differences were also noted, including enrichment of extracellular matrix (ECM) processes specifically in the airflow obstruction study. Network analysis of the ECM module implicated a candidate gene, matrix metalloproteinase 10 (MMP10), as a putative disease target. We used a knockout mouse model to functionally validate MMP10's role in influencing lung's susceptibility to cigarette smoke-induced emphysema. By integrating pathway analysis with population-based genomics, we unraveled biologic processes underlying pulmonary function traits and identified a candidate gene for obstructive lung disease.
Assuntos
Obstrução das Vias Respiratórias/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Pulmão/fisiopatologia , Polimorfismo de Nucleotídeo Único , Obstrução das Vias Respiratórias/fisiopatologia , Animais , Proliferação de Células , Genômica , Humanos , Sistema Imunitário , Masculino , Redes e Vias Metabólicas , Camundongos , Fenótipo , Transdução de Sinais , População Branca/genéticaRESUMO
RATIONALE: Syndecan-1 is a cell surface heparan sulfate proteoglycan primarily expressed in the lung epithelium. Because the influenza virus is tropic to the airway epithelium, we investigated the role of syndecan-1 in influenza infection. OBJECTIVES: To determine the mechanism by which syndecan-1 regulates the lung mucosal response to influenza infection. METHODS: Wild-type (WT) and Sdc1(-/-) mice were infected with a H1N1 virus (PR8) as an experimental model of influenza infection. Human and murine airway epithelial cell cultures were also infected with PR8 to study the mechanism by which syndecan-1 regulates the inflammatory response. MEASUREMENT AND MAIN RESULTS: We found worsened outcomes and lung injury in Sdc1(-/-) mice compared with WT mice after influenza infection. Our data demonstrated that syndecan-1 suppresses bronchial epithelial apoptosis during influenza infection to limit widespread lung inflammation. Furthermore, we determined that syndecan-1 attenuated apoptosis by crosstalking with c-Met to potentiate its cytoprotective signals in airway epithelial cells during influenza infection. CONCLUSIONS: Our work shows that cell-associated syndecan-1 has an important role in regulating lung injury. Our findings demonstrate a novel mechanism in which cell membrane-associated syndecan-1 regulates the innate immune response to influenza infection by facilitating cytoprotective signals through c-Met signaling to limit bronchial epithelial apoptosis, thereby attenuating lung injury and inflammation.
Assuntos
Apoptose/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Lesão Pulmonar/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Transdução de Sinais/imunologia , Sindecana-1/farmacologia , Animais , Modelos Animais de Doenças , Células Epiteliais/imunologia , Humanos , Imunidade Inata/imunologia , Pulmão/imunologia , Lesão Pulmonar/imunologia , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Sindecana-1/imunologiaRESUMO
Tissue inhibitor of metalloproteinases-3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3(-/-) mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3(-/-) mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow-derived macrophages (BMDMs) from WT and Timp3(-/-) mice revealed that Timp3(-/-) BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3(-/-) BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3(-/-) BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3(-/-) M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand-induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3(-/-) M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells.
Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Pneumonia/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Diferenciação Celular , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genótipo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Fenótipo , Pneumonia/genética , Pneumonia/imunologia , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-3/deficiência , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
RESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
Assuntos
Integrinas , Fibrose Pulmonar , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Modelos Animais de DoençasRESUMO
Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) represent serious health burdens because of both the tissue-damaging disease itself and an elevated risk of colon cancer. The increased expression of many members of the matrix metalloproteinase (MMP) family of enzymes that occurs in colitis has long been associated with the destructive nature of the disease. Recent findings in cancer and other MMP-associated diseases, however, led us to question whether MMPs are indeed detrimental in the setting of colitis. Here, we focus on a single MMP family member, MMP10, and assess its role in a murine model of colonic tissue damage induced by dextran sulfate sodium (DSS) treatment. Using mice genetically deficient for MMP10, we find that absence of this enzyme leads to significantly worse disease scores and failure to resolve inflammation even after extended recovery periods. We show that MMP10 is produced predominantly by infiltrating myeloid cells in both murine and human colitis. Through bone marrow transplant experiments, we confirm that bone marrow-derived MMP10 contributes to colitis severity. Mice lacking MMP10 have a significantly higher propensity for development of dysplastic lesions in the colon after two rounds of DSS exposure. Thus, we conclude that MMP10 is required for resolution of DSS-induced colonic damage, and in its absence, chronic inflammation and ultimately dysplasia occurs.
Assuntos
Colite Ulcerativa/enzimologia , Colo/enzimologia , Colo/patologia , Metaloproteinase 10 da Matriz/deficiência , Animais , Medula Óssea/enzimologia , Transplante de Medula Óssea , Linhagem Celular , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/química , Citocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Histocitoquímica , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/imunologia , Leucócitos/metabolismo , Masculino , Metaloproteinase 10 da Matriz/genética , Metaloproteinase 10 da Matriz/imunologia , Metaloproteinase 10 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Several members of the matrix metalloproteinase (MMP) family function in various processes of innate immunity, particularly in controlling leukocyte influx. Epilysin (MMP-28) is expressed in numerous tissues and, in adult mice, it has the highest expression in lung, where it is detected in bronchial epithelial cells (Clara cells). Epilysin is also expressed by bone marrow-derived macrophages, but not by alveolar macrophages, suggesting that its expression by macrophages is dependent on localization and differentiation. To assess the role of this MMP, we generated epilysin-null (Mmp28(-/-)) mice. Although epilysin is constitutively expressed in normal tissues, Mmp28(-/-) mice have no overt phenotype. However, using a murine model of Pseudomonas aeruginosa pneumonia, we found that Mmp28(-/-) mice had an early increase in macrophage recruitment into the lungs, as well as enhanced bacterial clearance and reduced pulmonary neutrophilia, which we predicted were due to accelerated macrophage influx. Macrophage depletion in WT and Mmp28(-/-) mice confirmed a role for macrophages in clearing P. aeruginosa and regulating neutrophil recruitment. Furthermore, we observed that macrophages derived from Mmp28(-/-) mice migrated faster than did wild-type cells to bronchoalveolar lavage fluid from P. aeruginosa-treated mice of either genotype. These observations indicate that epilysin functions as an intrinsic negative regulator of macrophage recruitment by retarding the chemotaxis of these cells.
Assuntos
Inibição de Migração Celular/imunologia , Quimiotaxia de Leucócito/imunologia , Macrófagos Alveolares/imunologia , Metaloproteinases da Matriz Secretadas/fisiologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Inibição de Migração Celular/genética , Células Cultivadas , Quimiotaxia de Leucócito/genética , Feminino , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/patologia , Masculino , Metaloproteinases da Matriz Secretadas/deficiência , Metaloproteinases da Matriz Secretadas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia Bacteriana/enzimologia , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/patologia , Fatores de TempoRESUMO
This study demonstrates that initiation of the CFTR modulator ivacaftor in people with cystic fibrosis and susceptible CFTR mutations causes an acute reduction in blood monocyte sensitivity to the key proinflammatory cytokine IFN-γ http://bit.ly/2TeI6LG.
RESUMO
BACKGROUND: CFTR modulators decrease some etiologies of CF airway inflammation; however, data indicate that non-resolving airway infection and inflammation persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation in the CFTR modulator era require better understanding of cellular contributions to chronic CF airway disease, and how inflammatory cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, represent intriguing cellular targets for focused therapies. This therapeutic approach, however, requires a more detailed knowledge of CF monocyte cellular programming and phenotypes. MATERIAL AND METHODS: In order to characterize the inflammatory phenotype of CF monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq) followed by pathway-based analyses. Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. RESULTS: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional programs were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including biological modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. CONCLUSIONS: Our results demonstrate that ivacaftor causes acute changes in blood monocyte transcriptional profiles and plasma chemokines, and suggest that increased monocyte inflammatory signals and changes in myeloid cell trafficking may contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of circulating blood monocytes in CF subjects treated with a CFTR modulator.
RESUMO
Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (matrix metalloproteinase 7 [MMP-7]) and stromelysin-2 (MMP-10), two MMPs induced by acute P. aeruginosa pulmonary infection. Extraction of differential gene expression (EDGE) analysis of gene expression changes in P. aeruginosa-infected organotypic tracheal epithelial cell cultures from wild-type, Mmp7-/-, and Mmp10-/- mice identified 2,091 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to Pseudomonas infection and show that a global genomics strategy can be used to assess MMP function.
Assuntos
Regulação da Expressão Gênica/fisiologia , Metaloproteinase 10 da Matriz/fisiologia , Metaloproteinase 7 da Matriz/fisiologia , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Animais , Indução Enzimática , Masculino , Metaloproteinase 10 da Matriz/biossíntese , Metaloproteinase 10 da Matriz/genética , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Mucosa Respiratória/enzimologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiologia , Transcrição GênicaRESUMO
Multiwalled carbon nanotubes (MWCNTs) are nanomaterials composed of multiple layers of graphene cylinders with unique properties that make them valuable for a number of industries. However, rising global production has led to concerns regarding potential occupational exposures to them as raw materials during handling. This is especially true for long MWCNT fibers, whose aspect ratio has been posited to initiate pathology similar to that of asbestos. Matrix metalloproteinases (MMPs) are a class of extracellular endopeptidases that control various processes related to tissue repair, inflammation, and more. Stromelysin-2 (MMP-10) has roles in modulating macrophage activation and function, and hence, we used an MMP-10 null (Mmp10-/-) mouse model to assess its role in controlling lung responses to inhaled long MWCNTs. Oropharyngeal aspiration of long MWCNTs (80 µg/mouse) by wild-type mice induced expression of Mmp10 mRNA, which was accompanied by a robust inflammatory response characterized by elevated expression of Tnfa, Il6, and Il1b. In Mmp10-/- mice, we found that absence of MMP-10 led to impaired pulmonary clearance of MWCNTs and reduced macrophage cell survival. Exposure of wild-type bone marrow-derived macrophages (BMDMs) and alveolar macrophages to MWCNTs caused a rapid, dose-dependent upregulation of Mmp10 mRNA expression, which was accompanied by expression of pro-inflammatory products (Il6 and Il1b). These products were further enhanced in Mmp10-/- macrophages, resulting in increased caspase-3-dependent cell death compared with wild-type cells. These findings indicate that MMP-10 facilitates the clearance of MWCNTs and moderates the pro-inflammatory response of exposed alveolar and infiltrated macrophages.
Assuntos
Inflamação/enzimologia , Pulmão/enzimologia , Pulmão/patologia , Metaloproteinase 10 da Matriz/metabolismo , Nanotubos de Carbono/toxicidade , Animais , Lavagem Broncoalveolar , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos Endogâmicos C57BL , Pneumonia/patologiaRESUMO
Matrix metalloproteinase-10 (MMP-10) is expressed by macrophages and epithelium in response to injury, but its functions in wound repair are unknown. We observed increased collagen deposition and skin stiffness in Mmp10(-/-) wounds, with no difference in collagen expression or reepithelialization. Increased collagen deposition in Mmp10(-/-) wounds was accompanied by less collagenolytic activity and reduced expression of specific metallocollagenases, particularly MMP-8 and MMP-13, where MMP-13 was the key collagenase. Ablation and adoptive transfer approaches and cell-based models demonstrated that the MMP-10-dependent collagenolytic activity was a product of alternatively activated (M2) resident macrophages. These data demonstrate a critical role for macrophage MMP-10 in controlling the tissue remodeling activity of macrophages and moderating scar formation during wound repair.
Assuntos
Colagenases/metabolismo , Metaloproteinase 10 da Matriz/metabolismo , Pele/metabolismo , Ferimentos e Lesões/enzimologia , Análise de Variância , Animais , Biópsia por Agulha , Células Cultivadas , Cicatriz/prevenção & controle , Modelos Animais de Doenças , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Masculino , Metaloproteinase 8 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Regeneração/fisiologia , Sensibilidade e Especificidade , Cicatrização/fisiologia , Ferimentos e Lesões/patologiaRESUMO
Members of the MMP family function in various processes of innate immunity, particularly in controlling important steps in leukocyte trafficking and activation. MMP28 (epilysin) is a member of this family of proteinases, and we have found that MMP28 is expressed by macrophages and regulates their recruitment to the lung. We hypothesized that MMP28 regulates other key macrophage responses, such as macrophage polarization. Furthermore, we hypothesized that these MMP28-dependent changes in macrophage polarization would alter fibrotic responses in the lung. We examined the gene expression changes in WT and Mmp28-/- BMDMs, stimulated with LPS or IL-4/IL-13 to promote M1 and M2 cells, respectively. We also collected macrophages from the lungs of Pseudomonas aeruginosa-exposed WT and Mmp28-/- mice to evaluate changes in macrophage polarization. Lastly, we evaluated the macrophage polarization phenotypes during bleomycin-induced pulmonary fibrosis in WT and Mmp28-/- mice and assessed mice for differences in weight loss and total collagen levels. We found that MMP28 dampens proinflammatory macrophage function and promots M2 programming. In both in vivo models, we found deficits in M2 polarization in Mmp28-/- mice. In bleomycin-induced lung injury, these changes were associated with reduced fibrosis. MMP28 is an important regulator of macrophage polarization, promoting M2 function. Loss of MMP28 results in reduced M2 polarization and protection from bleomycin-induced fibrosis. These findings highlight a novel role for MMP28 in macrophage biology and pulmonary disease.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , Metaloproteinases da Matriz Secretadas/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologia , Animais , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Citocinas/farmacologia , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Receptores Toll-Like/antagonistas & inibidoresRESUMO
This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2) facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h) and down-regulated later (24 h). Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 (-/-) bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 (-/-) cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.
Assuntos
Movimento Celular , Regulação Enzimológica da Expressão Gênica , Macrófagos/citologia , Macrófagos/imunologia , Metaloproteinase 10 da Matriz/genética , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Metaloproteinase 10 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
As their name implies, matrix metalloproteinases (MMPs) are thought to be responsible for the turnover of connective tissue proteins, a function that is indeed performed by some family members. However, matrix degradation is possibly not the predominant function of these enzymes. Several studies have demonstrated that MMPs also act on a variety of non-matrix extracellular proteins, such as cytokines, chemokines, receptors, junctional proteins, and antimicrobial peptides, to mediate a wide range of biological processes, such as repair, immunity, and angiogenesis. Our understanding of the many, diverse and, at times, unexpected functions of MMPs largely arose from the use of gene-targeted mice. In this chapter, we discuss the phenotypes of some MMP-deficient and TIMP-null mice and strategies and pitfalls in targeted mutagenesis.