RESUMO
Sarcoidosis is a chronic disease with unknown etiology and pathophysiology, characterized by granuloma formation. Matrix Metalloproteinase-12 (MMP12) is an elastase implicated in active granulomatous sarcoidosis. Previously, we reported that oropharyngeal instillation of multiwall carbon nanotubes (MWCNT) into C57Bl/6 mice induced sarcoid-like granulomas and upregulation of MMP12. When Mmp12 knock-out (KO) mice were instilled with MWCNT, granuloma formation occurred 10 days post-instillation but subsequently resolved at 60 days. Thus, we concluded that MMP12 was essential to granuloma persistence. The aim of the current study was to identify potential mechanisms of granuloma resolution in Mmp12KO mice. Strikingly, an M2 macrophage phenotype was present in Mmp12KO but not in C57Bl/6 mice. Between 10 and 60 days, macrophage populations in MWCNT-instilled Mmp12KO mice demonstrated an M2c to M2a phenotypic shift, with elevations in levels of IL-13, an M2 subtype-regulating factor. Furthermore, the M2 inducer, Apolipoprotein E (ApoE), and Matrix Metalloproteinase-14 (MMP14), a promoter of collagen degradation, were upregulated in 60-day MWCNT-instilled Mmp12KO mice. In conclusion, alveolar macrophages express two M2 phenotypes in Mmp12KO mice: M2c at 10 days when granulomas form, and M2a at 60 days when granulomas are resolving. Findings suggest that granuloma resolution in 60-day Mmp12KO mice requires an M2a macrophage phenotype.
Assuntos
Granuloma/imunologia , Pneumopatias/imunologia , Macrófagos Alveolares/imunologia , Metaloproteinase 12 da Matriz/genética , Animais , Granuloma/metabolismo , Pneumopatias/metabolismo , Camundongos , Camundongos Knockout , Nanotubos de CarbonoRESUMO
Poorly soluble environmental antigens, including carbon pollutants, are thought to play a role in the incidence of human sarcoidosis, a chronic inflammatory granulomatous disease of unknown causation. Currently, engineered carbon products such as multiwall carbon nanotubes (MWCNT) are manufactured commercially and have been shown to elicit acute and chronic inflammatory responses in experimental animals, including the production of granulomas or fibrosis. Several years ago, we hypothesized that constructing an experimental model of chronic granulomatosis resembling that associated with sarcoidosis might be achieved by oropharyngeal instillation of MWCNT into mice. This review summarizes the results of our efforts to define mechanisms of granuloma formation and identify potential therapeutic targets for sarcoidosis. Evidence is presented linking findings from the murine MWCNT granuloma model to sarcoidosis pathophysiology. As our goal was to determine what pulmonary inflammatory pathways might be involved, we utilized mice of knock-out (KO) backgrounds which corresponded to deficiencies noted in sarcoidosis patients. A primary example of this approach was to study mice with a myeloid-specific knock-out of the lipid-regulated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) which is strikingly depressed in sarcoidosis. Among the major findings associated with PPARγ KO mice compared to wild-type were: (1) exacerbation of granulomatous and fibrotic histopathology in response to MWCNT; (2) elevation of inflammatory mediators; and (3) pulmonary retention of a potentially antigenic ESAT-6 peptide co-instilled with MWCNT. In line with these data, we also observed that activation of PPARγ in wild-type mice by the PPARγ-specific ligand, rosiglitazone, significantly reduced both pulmonary granuloma and inflammatory mediator production. Similarly, recognition of a deficiency of ATP-binding cassette (ABC) lipid transporter ABCG1 in sarcoidosis led us to study MWCNT instillation in myeloid-specific ABCG1 KO mice. As anticipated, ABCG1 deficiency was associated with larger granulomas and increased levels of inflammatory mediators. Finally, a transcriptional survey of alveolar macrophages from MWCNT-instilled wild-type mice and human sarcoidosis patients revealed several common themes. One of the most prominent mediators identified in both human and mouse transcriptomic analyses was MMP12. Studies with MMP12 KO mice revealed similar acute reactions to those in wild-type but at chronic time points where wild-type maintained granulomatous disease, resolution occurred with MMP12 KO mice suggesting MMP12 is necessary for granuloma progression. In conclusion, these studies suggest that the MWCNT granuloma model has relevance to human sarcoidosis study, particularly with respect to immune-specific pathways.
Assuntos
Modelos Animais de Doenças , Granuloma , Nanotubos de Carbono , Sarcoidose , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , PPAR gama/metabolismoRESUMO
The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-ß) expression in lungs, together with an increased expression of TGF-ß related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin ß3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.
Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose/fisiologia , Líquido da Lavagem Broncoalveolar/citologia , Granuloma/induzido quimicamente , Granuloma/metabolismo , Nanotubos de Carbono/efeitos adversos , Fagocitose/fisiologia , Animais , Lavagem Broncoalveolar/métodos , Modelos Animais de Doenças , Doença Granulomatosa Crônica/metabolismo , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/metabolismo , Fibrose Pulmonar/metabolismo , Sarcoidose Pulmonar/metabolismoRESUMO
Background: Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation. Methods: C57BL/6 (wildtype) and Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1). Results: Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days. Conclusions: The striking reduction of granuloma formation at day 60 in Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.
Assuntos
Granuloma/imunologia , Macrófagos Alveolares/imunologia , Metaloproteinase 12 da Matriz/imunologia , Nanotubos de Carbono/efeitos adversos , Sarcoidose Pulmonar/imunologia , Animais , Granuloma/induzido quimicamente , Granuloma/genética , Granuloma/patologia , Macrófagos Alveolares/patologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Knockout , Sarcoidose Pulmonar/induzido quimicamente , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/patologiaRESUMO
Human exposure to carbon nanotubes (CNT) has been associated with the development of pulmonary sarcoid-like granulomatous disease. Our previous studies demonstrated that multi-walled carbon nanotubes (MWCNT) induced chronic pulmonary granulomatous inflammation in mice. Granuloma formation was accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) and disrupted intracellular lipid homeostasis in alveolar macrophages. Others have shown that PPARγ activation increases mitochondrial fatty acid oxidation (FAO) to reduce free fatty acid accumulation. Hence, we hypothesized that the disrupted lipid metabolism suppresses mitochondrial FAO. To test our hypothesis, C57BL/6 J mice were instilled by an oropharyngeal route with 100 µg MWCNT freshly suspended in 35 % Infasurf. Control sham mice received vehicle alone. Sixty days following instillation, mitochondrial FAO was measured in permeabilized bronchoalveolar lavage (BAL) cells. MWCNT instillation reduced the mitochondrial oxygen consumption rate of BAL cells in the presence of palmitoyl-carnitine as mitochondrial fuel. MWCNT also reduced mRNA expression of mitochondrial genes regulating FAO, carnitine palmitoyl transferase-1 (CPT1), carnitine palmitoyl transferase-2 (CPT2), hydroxyacyl-CoA dehydrogenase subunit beta (HADHB), and PPARγ coactivator 1 alpha (PPARGC1A). Importantly, both oxidative stress and apoptosis in alveolar macrophages and lung tissues of MWCNT-instilled mice were increased. Because macrophage PPARγ expression has been reported to be controlled by miR-27b which is known to induce oxidative stress and apoptosis, we measured the expression of miR-27b. Results indicated elevated levels in alveolar macrophages from MWCNT-instilled mice compared to controls. Given that inhibition of FAO and apoptosis are linked to M1 and M2 macrophage activation, respectively, the expression of both M1 and M2 key indicator genes were measured. Interestingly, results showed that both M1 and M2 phenotypes of alveolar macrophages were activated in MWCNT-instilled mice. In conclusion, alveolar macrophages of MWCNT-instilled mice had increased miR-27b expression, which may reduce the expression of PPARγ resulting in attenuation of FAO. This reduction in FAO may lead to activation of M1 macrophages. The upregulation of miR-27b may also induce apoptosis, which in turn can cause M2 activation of alveolar macrophages. These observations indicate a possible role of miR-27b in impaired mitochondrial function in the chronic activation of alveolar macrophages by MWCNT and the development of chronic pulmonary granulomatous inflammation.
Assuntos
Doença Granulomatosa Crônica/induzido quimicamente , Pneumopatias/induzido quimicamente , Macrófagos Alveolares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Animais , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologiaRESUMO
BACKGROUND: The pathological consequences of interaction between environmental carbon pollutants and microbial antigens have not been fully explored. We developed a murine model of multi-wall carbon nanotube (MWCNT)-elicited granulomatous disease which bears a striking resemblance to sarcoidosis, a human granulomatous disease. Because of reports describing lymphocyte reactivity to mycobacterial antigens in sarcoidosis patients, we hypothesized that addition of mycobacterial antigen (ESAT-6) to MWCNT might elicit activation in T cells. METHODS: Macrophage-specific peroxisome-proliferator-activated receptor gamma (PPARγ) knock out (KO) mice were studied along with wild-type mice because our previous report indicated PPARγ deficiency in sarcoidosis alveolar macrophages. MWCNT+ESAT-6 were instilled into mice. Controls received vehicle (surfactant-PBS) or ESAT-6 and were evaluated 60 days post-instillation. As noted in our recent publication, lung tissues from PPARγ KO mice instilled with MWCNT+ESAT-6 yielded more intensive pathophysiology, with elevated fibrosis. RESULTS: Inspection of mediastinal lymph nodes (MLN) revealed no granulomas but deposition of MWCNT. MLN cell counts were higher in PPARγ KO than in wild-type instilled with MWCNT+ESAT-6. Moreover, the CD4:CD8 T cell ratio, a major clinical metric for human disease, was increased in PPARγ KO mice. Bronchoalveolar lavage (BAL) cells from PPARγ KO mice instilled with MWCNT+ESAT-6 displayed increased Th17 cell markers (RORγt, IL-17A, CCR6) which associate with elevated fibrosis. CONCLUSION: These findings suggest that PPARγ deficiency in macrophages may promote ESAT-6-associated T cell activation in the lung, and that the MWCNT+ESAT-6 model may offer new insights into pathways of lymphocyte-mediated sarcoidosis histopathology.
Assuntos
Anticorpos Monoclonais/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Paraproteinemias/diagnóstico , Paraproteinemias/imunologia , Proteinose Alveolar Pulmonar/tratamento farmacológico , Anticorpos Monoclonais/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Biópsia , Medula Óssea/patologia , Cromatografia Líquida , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Cadeias gama de Imunoglobulina/sangue , Cadeias gama de Imunoglobulina/imunologia , Masculino , Gamopatia Monoclonal de Significância Indeterminada/sangue , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Paraproteinemias/sangue , Proteinose Alveolar Pulmonar/diagnóstico , Proteinose Alveolar Pulmonar/etiologia , Espectrometria de Massas em Tandem , Tomografia Computadorizada por Raios XRESUMO
Pulmonary granuloma formation is a complex and poorly understood response to inhaled pathogens and particulate matter. To explore the mechanisms of pulmonary granuloma formation and maintenance, our laboratory has developed a multiwall carbon nanotube (MWCNT)-induced murine model of chronic granulomatous inflammation. We have demonstrated that the MWCNT model closely mimics pulmonary sarcoidosis pathophysiology, including the deficiency of alveolar macrophage ATP-binding cassette (ABC) lipid transporters ABCA1 and ABCG1. We hypothesized that deficiency of alveolar macrophage ABCA1 and ABCG1 would promote pulmonary granuloma formation and inflammation. To test this hypothesis, the effects of MWCNT instillation were evaluated in ABCA1, ABCG1, and ABCA1/ABCG1 myeloid-specific knockout (KO) mice. Histological examination revealed significantly larger pulmonary granulomas in ABCG1-KO and ABCA1/ABCG1 double-KO animals when compared with wild-type animals. Evaluation of BAL cells indicated increased expression of CCL2 and osteopontin, genes shown to be involved in the formation and maintenance of pulmonary granulomas. Single deficiency of alveolar macrophage ABCA1 did not affect MWCNT-induced granuloma formation or proinflammatory gene expression. These observations indicate that the deficiency of alveolar macrophage ABCG1 promotes pulmonary granulomatous inflammation and that this is augmented by additional deletion of ABCA1.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Inflamação/metabolismo , Macrófagos Alveolares/metabolismo , Sarcoidose Pulmonar/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Granuloma/metabolismo , Pulmão/metabolismo , Camundongos Knockout , Pneumonia/metabolismoRESUMO
We established a murine model of multiwall carbon nanotube (MWCNT)-elicited chronic granulomatous disease that bears similarities to human sarcoidosis pathology, including alveolar macrophage deficiency of peroxisome proliferator-activated receptor γ (PPARγ). Because lymphocyte reactivity to mycobacterial antigens has been reported in sarcoidosis, we hypothesized that addition of mycobacterial ESAT-6 (early secreted antigenic target protein 6) to MWCNT might exacerbate pulmonary granulomatous pathology. MWCNTs with or without ESAT-6 peptide 14 were instilled by the oropharyngeal route into macrophage-specific PPARγ-knockout (KO) or wild-type mice. Control animals received PBS or ESAT-6. Lung tissues, BAL cells, and BAL fluid were evaluated 60 days after instillation. PPARγ-KO mice receiving MWCNT + ESAT-6 had increased granulomas and significantly elevated fibrosis (trichrome staining) compared with wild-type mice or PPARγ-KO mice that received only MWCNT. Immunostaining of lung tissues revealed elevated fibronectin and Siglec F expression on CD11c+ infiltrating alveolar macrophages in the presence of MWCNT + ESAT-6 compared with MWCNT alone. Analyses of BAL fluid proteins indicated increased levels of transforming growth factor (TGF)-ß and the TGF-ß pathway mediator IL-13 in PPARγ-KO mice that received MWCNT + ESAT-6 compared with wild-type or PPARγ-KO mice that received MWCNT. Similarly, mRNA levels of matrix metalloproteinase 9, another requisite factor for TGF-ß production, was elevated in PPARγ-KO mice by MWCNT + ESAT-6. Analysis of ESAT-6 in lung tissues by mass spectrometry revealed ESAT-6 retention in lung tissues of PPARγ-KO but not wild-type mice. These data indicate that PPARγ deficiency promotes pulmonary ESAT-6 retention, exacerbates macrophage responses to MWCNT + ESAT-6, and intensifies pulmonary fibrosis. The present findings suggest that the model may facilitate understanding of the effects of environmental factors on sarcoidosis-associated pulmonary fibrosis.
Assuntos
Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Macrófagos Alveolares/metabolismo , PPAR gama/deficiência , Fibrose Pulmonar/microbiologia , Sarcoidose Pulmonar/microbiologia , Animais , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar , Antígenos CD11/metabolismo , Modelos Animais de Doenças , Fibronectinas/metabolismo , Fibrose/metabolismo , Inflamação , Pulmão/patologia , Macrófagos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanotubos de Carbono/química , PPAR gama/genética , Fibrose Pulmonar/genética , Sarcoidose Pulmonar/patologiaRESUMO
Peroxisome proliferator activated receptor gamma (PPARγ), a ligand activated nuclear transcription factor, is constitutively expressed in alveolar macrophages of healthy individuals. PPARγ deficiencies have been noted in several lung diseases including the alveolar macrophages of pulmonary sarcoidosis patients. We have previously described a murine model of multiwall carbon nanotubes (MWCNT) induced pulmonary granulomatous inflammation which bears striking similarities to pulmonary sarcoidosis, including the deficiency of alveolar macrophage PPARγ. Further studies demonstrate alveolar macrophage PPARγ deficiency exacerbates MWCNT-induced pulmonary granulomas. Based on these observations we hypothesized that activation of PPARγ via administration of the PPARγ-specific ligand rosiglitazone would limit MWCNT-induced granuloma formation and promote PPARγ-dependent pathways. Results presented here show that rosiglitazone significantly limits the frequency and severity of MWCNT-induced pulmonary granulomas. Furthermore, rosiglitazone attenuates alveolar macrophage NF-κB activity and downregulates the expression of the pro-inflammatory mediators, CCL2 and osteopontin. PPARγ activation via rosiglitazone also prevents the MWCNT-induced deficiency of PPARγ-regulated ATP-binding cassette lipid transporter-G1 (ABCG1) expression. ABCG1 is crucial to pulmonary lipid homeostasis. ABCG1 deficiency results in lipid accumulation which promotes pro-inflammatory macrophage activation. Our results indicate that restoration of homeostatic ABCG1 levels by rosiglitazone correlates with both reduced pulmonary lipid accumulation, and decreased alveolar macrophage activation. These data confirm and further support our previous observations that PPARγ pathways are critical in regulating MWCNT-induced pulmonary granulomatous inflammation.
Assuntos
Granuloma/patologia , Pneumopatias/patologia , Pulmão/patologia , PPAR gama/metabolismo , Sarcoidose/patologia , Transdução de Sinais , Animais , Modelos Animais de Doenças , Dislipidemias/etiologia , Dislipidemias/genética , Dislipidemias/metabolismo , Dislipidemias/patologia , Regulação da Expressão Gênica , Granuloma/etiologia , Granuloma/genética , Granuloma/metabolismo , Pulmão/metabolismo , Pneumopatias/etiologia , Pneumopatias/genética , Pneumopatias/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/efeitos adversos , PPAR gama/agonistas , Sarcoidose/etiologia , Sarcoidose/genética , Sarcoidose/metabolismoRESUMO
Mohan A, Malur A, McPeek M, Barna BP, Schnapp LM, Thomassen MJ, Gharib SA. Transcriptional survey of alveolar macrophages in a murine model of chronic granulomatous inflammation reveals common themes with human sarcoidosis. Am J Physiol Lung Cell Mol Physiol 314: L617-L625, 2018. First published December 6, 2017; doi: 10.1152/ajplung.00289.2017 . To advance our understanding of the pathobiology of sarcoidosis, we developed a multiwall carbon nanotube (MWCNT)-based murine model that shows marked histological and inflammatory signal similarities to this disease. In this study, we compared the alveolar macrophage transcriptional signatures of our animal model with human sarcoidosis to identify overlapping molecular programs. Whole genome microarrays were used to assess gene expression of alveolar macrophages in six MWCNT-exposed and six control animals. The results were compared with the transcriptional profiles of alveolar immune cells in 15 sarcoidosis patients and 12 healthy humans. Rigorous statistical methods were used to identify differentially expressed genes. To better elucidate activated pathways, integrated network and gene set enrichment analysis (GSEA) was performed. We identified over 1,000 differentially expressed between control and MWCNT mice. Gene ontology functional analysis showed overrepresentation of processes primarily involved in immunity and inflammation in MCWNT mice. Applying GSEA to both mouse and human samples revealed upregulation of 92 gene sets in MWCNT mice and 142 gene sets in sarcoidosis patients. Commonly activated pathways in both MWCNT mice and sarcoidosis included adaptive immunity, T-cell signaling, IL-12/IL-17 signaling, and oxidative phosphorylation. Differences in gene set enrichment between MWCNT mice and sarcoidosis patients were also observed. We applied network analysis to differentially expressed genes common between the MWCNT model and sarcoidosis to identify key drivers of disease. In conclusion, an integrated network and transcriptomics approach revealed substantial functional similarities between a murine model and human sarcoidosis particularly with respect to activation of immune-specific pathways.
Assuntos
Modelos Animais de Doenças , Perfilação da Expressão Gênica , Doença Granulomatosa Crônica/metabolismo , Inflamação/metabolismo , Macrófagos Alveolares/metabolismo , Sarcoidose/metabolismo , Animais , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/química , Sarcoidose/genética , Sarcoidose/patologia , Transcrição GênicaRESUMO
Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.
Assuntos
Fígado Gorduroso/metabolismo , Fígado/metabolismo , Macrófagos Alveolares/metabolismo , Fosfatidilcolinas/metabolismo , Proteinose Alveolar Pulmonar/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Fígado Gorduroso/complicações , Fígado Gorduroso/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética , Fosfatidilcolinas/genética , Proteinose Alveolar Pulmonar/etiologia , Proteinose Alveolar Pulmonar/genéticaRESUMO
BACKGROUND: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease. METHODS: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. CONTROLS: To better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles. RESULTS: Sarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis. CONCLUSIONS: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Regulação da Expressão Gênica/genética , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/metabolismo , Adulto , Idoso , Broncoscopia , Contagem de Células , Citocinas/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Humanos , Imunidade/genética , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/genética , RNA/biossíntese , RNA/isolamento & purificação , Sarcoidose Pulmonar/imunologia , Fumar/genéticaRESUMO
We established a murine model of multiwall carbon nanotube (MWCNT)-induced chronic granulomatous disease, which resembles human sarcoidosis pathology. At 60 days after oropharyngeal MWCNT instillation, bronchoalveolar lavage (BAL) cells from wild-type mice exhibit an M1 phenotype with elevated proinflammatory cytokines and reduced peroxisome proliferator-activated receptor γ (PPARγ)-characteristics also present in human sarcoidosis. Based upon MWCNT-associated PPARγ deficiency, we hypothesized that the PPARγ target gene, ATP-binding cassette (ABC) G1, a lipid transporter with antiinflammatory properties, might also be repressed. Results after MWCNT instillation indicated significantly repressed ABCG1, but, surprisingly, lipid transporter ABCA1 was also repressed, suggesting a possible second pathway. Exploration of potential regulators revealed that microRNA (miR)-33, a lipid transporter regulator, was strikingly elevated (13.9 fold) in BAL cells from MWCNT-instilled mice but not sham control mice. Elevated miR-33 was also detected in murine granulomatous lung tissue. In vitro studies confirmed that lentivirus-miR-33 overexpression repressed both ABCA1 and ABCG1 (but not PPARγ) in cultured murine alveolar macrophages. BAL cells of patients with sarcoidosis also displayed elevated miR-33 together with reduced ABCA1 and ABCG1 messenger RNA and protein compared with healthy control subjects. Moreover, miR-33 was elevated within sarcoidosis granulomatous tissue. The findings suggest that alveolar macrophage miR-33 is up-regulated by proinflammatory cytokines and may perpetuate chronic inflammatory granulomatous disease by repressing antiinflammatory functions of ABCA1 and ABCG1 lipid transporters. The results also suggest two possible pathways for transporter dysregulation in granulomatous disease-one associated with intrinsic PPARγ status and the other with miR-33 up-regulation triggered by environmental challenges, such as MWCNT.
Assuntos
Doença Granulomatosa Crônica/induzido quimicamente , Doença Granulomatosa Crônica/genética , MicroRNAs/metabolismo , Nanotubos de Carbono/efeitos adversos , Sarcoidose/genética , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Doença Granulomatosa Crônica/patologia , Humanos , Lipídeos/química , Macrófagos Alveolares/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
Recent studies suggest additive effects of environmental pollutants and microbial antigens on respiratory disease. We established a granuloma model in which instilled multiwall carbon nanotubes (MWCNT) elicit granulomatous pathology. We hypothesized that mycobacterial antigen ESAT-6, a T cell activator associated with tuberculosis and sarcoidosis, might alter pathology. Wild-type C57Bl/6 mice received MWCNT with or without ESAT-6 peptide. Controls received vehicle (surfactant-PBS) or ESAT-6 alone. Mice were evaluated 60 days later for granulomas, fibrosis, and bronchoalveolar lavage (BAL) cell expression of inflammatory mediators (CCL2, MMP-12, and Osteopontin). Results indicated increased granulomas, fibrosis, and inflammatory mediators in mice receiving the combination of MWCNT+ESAT-6 compared to MWCNT or vehicle alone. ESAT-6 alone showed no significant effect on these pathological endpoints. However, CD3 (+) lymphocyte infiltration of lung tissue increased with MWCNT+ESAT-6 versus MWCNT alone. Findings suggest that concurrent exposure to microbial antigen and MWCNT exacerbates chronic pulmonary disease.
RESUMO
Sarcoidosis, a chronic granulomatous disease of unknown cause, has been linked to several environmental risk factors, among which are some that may favor carbon nanotube formation. Using gene array data, we initially observed that bronchoalveolar lavage (BAL) cells from sarcoidosis patients displayed elevated mRNA of the transcription factor, Twist1, among many M1-associated genes compared to healthy controls. Based on this observation we hypothesized that Twist1 mRNA and protein expression might become elevated in alveolar macrophages from animals bearing granulomas induced by carbon nanotube instillation. To address this hypothesis, wild-type and macrophage-specific peroxisome proliferator-activated receptor gamma (PPARγ) knock out mice were given oropharyngeal instillation of multiwall carbon nanotubes (MWCNT). BAL cells obtained 60 days later exhibited significantly elevated Twist1 mRNA expression in granuloma-bearing wild-type or PPARγ knock out alveolar macrophages compared to sham controls. Overall, Twist1 expression levels in PPARγ knock out mice were higher than those of wild-type. Concurrently, BAL cells obtained from sarcoidosis patients and healthy controls validated gene array data: qPCR and protein analysis showed significantly elevated Twist1 in sarcoidosis compared to healthy controls. In vitro studies of alveolar macrophages from healthy controls indicated that Twist1 was inducible by classical (M1) macrophage activation stimuli (LPS, TNFα) but not by IL-4, an inducer of alternative (M2) macrophage activation. Findings suggest that Twist1 represents a PPARγ-sensitive alveolar macrophage M1 biomarker which is induced by inflammatory granulomatous disease in the MWCNT model and in human sarcoidosis.
Assuntos
Macrófagos Alveolares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adulto , Animais , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Ativação de Macrófagos , Macrófagos Alveolares/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , PPAR gama/deficiência , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Sarcoidose Pulmonar/induzido quimicamente , Sarcoidose Pulmonar/metabolismo , Sarcoidose Pulmonar/patologia , Proteína 1 Relacionada a Twist/genética , Regulação para CimaRESUMO
BACKGROUND: Activin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs in vitro. To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARγ). RESULTS: In sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNγ, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNγ mRNA was not elevated in PAP BAL cells. In vitro studies confirmed that IFNγ stimulated activin A in wild-type AMs while antibody to IFNγ reduced activin A in GM-CSF knockout AMs. Both IFNγ and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARγ compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNγ-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated. CONCLUSIONS: Data point to IFNγ as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Macrófagos Alveolares/metabolismo , Proteinose Alveolar Pulmonar/metabolismo , Ativinas/genética , Ativinas/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imuno-Histoquímica , Interferon gama/genética , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos Alveolares/classificação , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Proteinose Alveolar Pulmonar/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although granulomatous inflammation is a central feature of many disease processes, cellular mechanisms of granuloma formation and persistence are poorly understood. Carbon nanoparticles, which can be products of manufacture or the environment, have been associated with granulomatous disease. This paper utilizes a previously described carbon nanoparticle granuloma model to address the issue of whether peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear transcription factor and negative regulator of inflammatory cytokines might play a role in granulomatous lung disease. PPARγ is constitutively expressed in alveolar macrophages from healthy individuals but is depressed in alveolar macrophages of patients with sarcoidosis, a prototypical granulomatous disease. Our previous study of macrophage-specific PPARγ KO mice had revealed an intrinsically inflammatory pulmonary environment with an elevated pro-inflammatory cytokines profile as compared to wild-type mice. Based on such observations we hypothesized that PPARγ expression would be repressed in alveolar macrophages from animals bearing granulomas induced by MWCNT instillation. METHODS: Wild-type C57Bl/6 and macrophage-specific PPARγ KO mice received oropharyngeal instillations of multiwall carbon nanotubes (MWCNT) (100 µg). Bronchoalveolar lavage (BAL) cells, BAL fluids, and lung tissues were obtained 60 days post-instillation for analysis of granuloma histology and pro-inflammatory cytokines (osteopontin, CCL2, and interferon gamma [IFN-γ] mRNA and protein expression. RESULTS: In wild-type mice, alveolar macrophage PPARγ expression and activity were significantly reduced in granuloma-bearing animals 60 days after MWCNT instillation. In macrophage-specific PPARγ KO mice, granuloma formation was more extensive than in wild-type at 60 days after MWCNT instillation. PPARγ KO mice also demonstrated elevated pro-inflammatory cytokine expression in lung tissue, laser-microdissected lung granulomas, and BAL cells/fluids, at 60 days post MWCNT exposure. CONCLUSIONS: Overall, data indicate that PPARγ deficiency promotes inflammation and granuloma formation, suggesting that PPARγ functions as a negative regulator of chronic granulomatous inflammation.
Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Granuloma do Sistema Respiratório/imunologia , Pulmão/imunologia , Nanotubos de Carbono , PPAR gama/imunologia , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Dysfunctional immune responses characterize sarcoidosis, but the status of cathelicidin, a potent immunoregulatory and antimicrobial molecule, has not been established in clinical disease activity. METHODS: Alveolar macrophage cathelicidin expression was determined in biopsy-proven sarcoidosis patients classified clinically as 'severe' (requiring systemic treatment) or 'non-severe' (never requiring treatment). Bronchoalveolar lavage (BAL) cells from sarcoidosis patients and healthy controls were analyzed for mRNA expression of cathelicidin, vitamin D receptor (VDR) and the VDR coactivator steroid receptor coactivator-3 (SRC3) by quantitative PCR. Cathelicidin-derived peptide LL-37 was determined by immunocytochemistry. Serum calcidiol (25-hydroxyvitamin D2; vitD2) and calcitriol (1,25-dihydroxyvitamin D3; vitD3) were quantified. RESULTS: The results indicated reduced BAL cell expression of cathelicidin and SRC3 in severe but not non-severe sarcoidosis compared to controls. Serum levels of biologically active vitD3 in both severe and non-severe patients were within the control range even though vitD2 levels in both groups were below the recommended level (30 ng/ml). Sarcoidosis and control alveolar macrophages were studied in vitro to determine cathelicidin responses to vitD3 and tumor necrosis factor-α (TNFα), a vitD3 antagonist elevated in active sarcoidosis. Alveolar macrophage cathelicidin was stimulated by vitD3 but repressed by TNFα, which also repressed SRC3. CONCLUSIONS: These findings suggest that TNFα-mediated repression of SRC3 contributes to alveolar macrophage cathelicidin deficiency in severe sarcoidosis despite healthy vitD3 levels. Deficiency of cathelicidin, a multifunctional regulator of immune cells and proinflammatory cytokines, may impede resolution of inflammation in the lungs of patients with severe sarcoidosis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/deficiência , Macrófagos Alveolares/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Sarcoidose Pulmonar/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Vitamina D/análogos & derivados , Adulto , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Coativador 3 de Receptor Nuclear/genética , Sarcoidose Pulmonar/metabolismo , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Vitamina D/metabolismo , Adulto Jovem , CatelicidinasRESUMO
BACKGROUND: Classically, activated macrophages in adipose tissue, liver, and muscle have been implicated in many conditions associated with obesity, including insulin resistance and the metabolic syndrome. Despite numerous pulmonary comorbidities and the sentinel role alveolar macrophages play in innate immunity and lung homeostasis, their activation status has not been examined in these patients. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) has been shown to be a negative regulator of inflammation in addition to regulating lipid and glucose metabolism. PPAR-γ is expressed constitutively in healthy alveolar macrophages and decreased on activation. We hypothesized that PPAR-γ would be downregulated in alveolar macrophages from obese patients with obstructive sleep apnea (OSA) in the absence of overt lung disease. METHODS: Alveolar macrophages were obtained by bronchoalveolar lavage from obese individuals with and without OSA and healthy controls. RESULTS: Data indicated that PPAR-γ functional activity was decreased by 48% in obese with OSA and 26% without OSA (P < .05). In obese patients with OSA, PPAR-γ mRNA was decreased 2-fold compared with controls (P < .05), whereas obese patients without OSA, it was not different. Regardless of OSA, alveolar macrophages of obese patients demonstrated increased interleukin-6 mRNA. CONCLUSION: These findings are consistent with the presence of classic macrophage activation and an inflammatory lung environment. Data from this study suggest that alveolar macrophage dysfunction becomes aggravated in OSA and may increase pulmonary disease susceptibility.