RESUMEN
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.
Asunto(s)
Integrinas , Fibrosis Pulmonar , Animales , Ratones , Membrana Celular , Microscopía por Crioelectrón , Modelos Animales de EnfermedadRESUMEN
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.
RESUMEN
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.
Asunto(s)
Lesión Pulmonar Aguda , MAP Quinasa Quinasa 2/metabolismo , Síndrome de Dificultad Respiratoria , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , MAP Quinasa Quinasa 2/genética , Ratones , Síndrome de Dificultad Respiratoria/genéticaRESUMEN
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.
Asunto(s)
Coxiella burnetii , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Fiebre Q , Animales , Ratones , Ratones Endogámicos C57BL , Fiebre Q/inmunologíaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Carcinogénesis/metabolismo , Exosomas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , Sindecano-1/metabolismo , Células A549 , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Proliferación Celular , Regulación hacia Abajo/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/patología , Ratones , MicroARNs/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba/genéticaRESUMEN
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.
Asunto(s)
Inflamación/enzimología , Pulmón/enzimología , Pulmón/patología , Metaloproteinasa 10 de la Matriz/metabolismo , Nanotubos de Carbono/toxicidad , Animales , Lavado Broncoalveolar , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Endocitosis/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Pulmón/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones Endogámicos C57BL , Neumonía/patologíaRESUMEN
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.
Asunto(s)
Inflamación/microbiología , Inflamación/virología , Inovirus/crecimiento & desarrollo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/virología , Pseudomonas aeruginosa/virología , Animales , Biopelículas/crecimiento & desarrollo , Fibrosis Quística/microbiología , Fibrosis Quística/virología , Pulmón/microbiología , Pulmón/virología , Macrófagos/microbiología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Fagocitosis/fisiologíaRESUMEN
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.
Asunto(s)
Fibrosis Quística/inmunología , Inflamación/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Metaloproteinasa 10 de la Matriz/inmunología , Traslado Adoptivo , Animales , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Pseudomonas/inmunologíaRESUMEN
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.
Asunto(s)
Apoptosis/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Lesión Pulmonar/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Transducción de Señal/inmunología , Sindecano-1/farmacología , Animales , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Humanos , Inmunidad Innata/inmunología , Pulmón/inmunología , Lesión Pulmonar/inmunología , Ratones , Proteínas Proto-Oncogénicas c-met/genética , Sindecano-1/inmunologíaRESUMEN
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.
Asunto(s)
Obstrucción de las Vías Aéreas/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Pulmón/fisiopatología , Polimorfismo de Nucleótido Simple , Obstrucción de las Vías Aéreas/fisiopatología , Animales , Proliferación Celular , Genómica , Humanos , Sistema Inmunológico , Masculino , Redes y Vías Metabólicas , Ratones , Fenotipo , Transducción de Señal , Población Blanca/genéticaRESUMEN
Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection.
Asunto(s)
Encéfalo/metabolismo , Pulmón/metabolismo , Infecciones por Pseudomonas/genética , ARN Mensajero/genética , Versicanos/genética , Animales , Encéfalo/ultraestructura , Embrión de Mamíferos , Desarrollo Embrionario/fisiología , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Pulmón/ultraestructura , Ratones , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Proteolisis , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/crecimiento & desarrollo , ARN Mensajero/metabolismo , Versicanos/metabolismoRESUMEN
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.
Asunto(s)
Colagenasas/metabolismo , Metaloproteinasa 10 de la Matriz/metabolismo , Piel/metabolismo , Heridas y Lesiones/enzimología , Análisis de Varianza , Animales , Biopsia con Aguja , Células Cultivadas , Cicatriz/prevención & control , Modelos Animales de Enfermedad , Epitelio/metabolismo , Femenino , Humanos , Inmunohistoquímica , Macrófagos/metabolismo , Masculino , Metaloproteinasa 8 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Regeneración/fisiología , Sensibilidad y Especificidad , Cicatrización de Heridas/fisiología , Heridas y Lesiones/patologíaRESUMEN
Cysteine-rich protein-61 (CYR61), also known as connective tissue growth factor, CYR61, and nephroblastoma overexpressed gene 1 (CCN1), is a heparin-binding protein member of the CCN family of matricellular proteins. Gene expression profiles showed that Cyr61 is upregulated in human acute lung injury (ALI), but its functional role is unclear. We hypothesized that CYR61 contributes to ALI in mice. First, we demonstrated that CYR61 expression increases after bleomycin-induced lung injury. We then used adenovirus-mediated gene transfer to determine whether CYR61 overexpression in the lungs was sufficient to cause ALI. Mice instilled with CYR61 adenovirus showed greater weight loss, increased bronchoalveolar lavage total neutrophil counts, increased protein concentrations, and increased mortality compared with mice instilled with empty-vector adenovirus. Immunohistochemical studies in lungs from humans with idiopathic pulmonary fibrosis revealed CYR61 expression on the luminal membrane of alveolar epithelial cells in areas of injury. We conclude that CYR61 is upregulated in ALI and that CYR61 overexpression exacerbates ALI in mice.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Proteína 61 Rica en Cisteína/metabolismo , Expresión Génica , Animales , Proteína 61 Rica en Cisteína/genética , Humanos , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Síndrome de Dificultad Respiratoria/metabolismoRESUMEN
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.
Asunto(s)
Macrófagos/inmunología , Macrófagos/metabolismo , Metaloproteinasas de la Matriz Secretadas/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Animales , Apoptosis/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Citocinas/farmacología , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/citología , Macrófagos Alveolares/citología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ratones , Ratones Noqueados , Monocitos/metabolismo , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Estrés Fisiológico/genética , Receptores Toll-Like/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Neumonía/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Animales , Diferenciación Celular , Citocinas/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Genotipo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Fenotipo , Neumonía/genética , Neumonía/inmunología , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-3/deficiencia , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
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.
Asunto(s)
Movimiento Celular , Regulación Enzimológica de la Expresión Génica , Macrófagos/citología , Macrófagos/inmunología , Metaloproteinasa 10 de la Matriz/genética , Animales , Células de la Médula Ósea/citología , Línea Celular , Movimiento Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Metaloproteinasa 10 de la Matriz/deficiencia , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Colitis Ulcerosa/enzimología , Colon/enzimología , Colon/patología , Metaloproteinasa 10 de la Matriz/deficiencia , Animales , Médula Ósea/enzimología , Trasplante de Médula Ósea , Línea Celular , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colon/química , Citocinas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Histocitoquímica , Humanos , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/inmunología , Leucocitos/metabolismo , Masculino , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/inmunología , Metaloproteinasa 10 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.