Inflammatory Pathways in Sarcoidosis.

Concepts regarding etiology and pathophysiology of sarcoidosis have changed remarkably within the past 5 years. Sarcoidosis is now viewed as a complex multi-causation disease related to a diverse collection of external environmental or infectious signals. It is generally accepted that the cause of sarcoidosis is unknown. Moreover, concepts of the inflammatory pathway have been modified by the realization that intrinsic genetic factors and innate immunity may modify adaptive immune responses to external triggers. With those potential regulatory pathways in mind, we will attempt to discuss the current understanding of the inflammatory response in sarcoidosis with emphasis on development of pulmonary granulomatous pathology. In that context, we will emphasize that both macrophages and T lymphocytes play key roles, with sometimes overlapping cytokine production (i.e., TNFα and IFN-γ) but also with unique mediators that influence the pathologic picture. Numerous studies have shown that in a sizable number of sarcoidosis patients, granulomas spontaneously resolve, usually within 3 years. Other sarcoidosis patients, however, may develop a chronic granulomatous disease which may subsequently lead to fibrosis. This chapter will outline our current understanding of inflammatory pathways in sarcoidosis which initiate and mediate granulomatous changes or onset of pulmonary fibrosis.

Studies in a Murine Granuloma Model of Instilled Carbon Nanotubes: Relevance to Sarcoidosis.

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.

The M2a Macrophage Phenotype Accompanies Pulmonary Granuloma Resolution in Mmp12 Knock-Out Mice Instilled with Multiwall Carbon Nanotubes.

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.

Alveolar Macrophage ABCG1 Deficiency Promotes Pulmonary Granulomatous Inflammation.

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.

Peroxisome Proliferator-activated Receptor-γ Deficiency Exacerbates Fibrotic Response to Mycobacteria Peptide in Murine Sarcoidosis Model.

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.

Transcriptional survey of alveolar macrophages in a murine model of chronic granulomatous inflammation reveals common themes with human sarcoidosis.

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.

PPAR-gamma pathways attenuate pulmonary granuloma formation in a carbon nanotube induced murine model of sarcoidosis.

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.

Elevated MicroRNA-33 in Sarcoidosis and a Carbon Nanotube Model of Chronic Granulomatous Disease.

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.

Sarcoidosis activates diverse transcriptional programs in bronchoalveolar lavage cells.

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.

Alveolar macrophages of GM-CSF knockout mice exhibit mixed M1 and M2 phenotypes.

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.

The role of PPARγ in carbon nanotube-elicited granulomatous lung inflammation.

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.

Carbon nanotube-induced pulmonary granulomatous disease: Twist1 and alveolar macrophage M1 activation.

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.

Rituximab therapy in pulmonary alveolar proteinosis improves alveolar macrophage lipid homeostasis.

RATIONALE: Pulmonary Alveolar Proteinosis (PAP) patients exhibit an acquired deficiency of biologically active granulocyte-macrophage colony stimulating factor (GM-CSF) attributable to GM-CSF specific autoantibodies. PAP alveolar macrophages are foamy, lipid-filled cells with impaired surfactant clearance and markedly reduced expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) and the PPARγ-regulated ATP binding cassette (ABC) lipid transporter, ABCG1. An open label proof of concept Phase II clinical trial was conducted in PAP patients using rituximab, a chimeric murine-human monoclonal antibody directed against B lymphocyte specific antigen CD20. Rituximab treatment decreased anti-GM-CSF antibody levels in bronchoalveolar lavage (BAL) fluid, and 7/9 patients completing the trial demonstrated clinical improvement as measured by arterial blood oxygenation. OBJECTIVES: This study sought to determine whether rituximab therapy would restore lipid metabolism in PAP alveolar macrophages. METHODS: BAL samples were collected from patients pre- and 6-months post-rituximab infusion for evaluation of mRNA and lipid changes. RESULTS: Mean PPARγ and ABCG1 mRNA expression increased 2.8 and 5.3-fold respectively (p ≤ 0.05) after treatment. Lysosomal phospholipase A2 (LPLA2) (a key enzyme in surfactant degradation) mRNA expression was severely deficient in PAP patients pre-treatment but increased 2.8-fold post-treatment. In supplemental animal studies, LPLA2 deficiency was verified in GM-CSF KO mice but was not present in macrophage-specific PPARγ KO mice compared to wild-type controls. Oil Red O intensity of PAP alveolar macrophages decreased after treatment, indicating reduced intracellular lipid while extracellular free cholesterol increased in BAL fluid. Furthermore, total protein and Surfactant protein A were significantly decreased in the BAL fluid post therapy. CONCLUSIONS: Reduction in GM-CSF autoantibodies by rituximab therapy improves alveolar macrophage lipid metabolism by increasing lipid transport and surfactant catabolism. Mechanisms may involve GM-CSF stimulation of alveolar macrophage ABCG1 and LPLA2 activities by distinct pathways.

Novel murine model of chronic granulomatous lung inflammation elicited by carbon nanotubes.

Lung granulomas are associated with numerous conditions, including inflammatory disorders, exposure to environmental pollutants, and infection. Osteopontin is a chemotactic cytokine produced by macrophages, and is implicated in extracellular matrix remodeling. Furthermore, osteopontin is up-regulated in granulomatous disease, and osteopontin null mice exhibit reduced granuloma formation. Animal models currently used to investigate chronic lung granulomatous inflammation bear a pathological resemblance, but lack the chronic nature of human granulomatous disease. Carbon nanoparticles are generated as byproducts of combustion. Interestingly, experimental exposures to carbon nanoparticles induce pulmonary granuloma-like lesions. However, the recruited cellular populations and extracellular matrix gene expression profiles within these lesions have not been explored. Because of the rapid resolution of granulomas in current animal models, the mechanisms responsible for persistence have been elusive. To overcome the limitations of previous models, we investigated whether a model using multiwall carbon nanoparticles would resemble chronic human lung granulomatous inflammation. We hypothesized that pulmonary exposure to multiwall carbon nanoparticles would induce granulomas, elicit a macrophage and T-cell response, and mimic other granulomatous disorders with an up-regulation of osteopontin. This model demonstrates: (1) granulomatous inflammation, with macrophage and T-cell infiltration; (2) resemblance to the chronicity of human granulomas, with persistence up to 90 days; and (3) a marked elevation of osteopontin, metalloproteinases, and cell adhesion molecules in granulomatous foci isolated by laser-capture microdissection and in alveolar macrophages from bronchoalveolar lavage. The establishment of such a model provides an important platform for mechanistic studies on the persistence of granuloma.

Restoration of PPARγ reverses lipid accumulation in alveolar macrophages of GM-CSF knockout mice.

Pulmonary alveolar proteinosis (PAP) is a lung disease characterized by a deficiency of functional granulocyte macrophage colony-stimulating factor (GM-CSF) resulting in surfactant accumulation and lipid-engorged alveolar macrophages. GM-CSF is a positive regulator of PPARγ that is constitutively expressed in healthy alveolar macrophages. We previously reported decreased PPARγ and ATP-binding cassette transporter G1 (ABCG1) levels in alveolar macrophages from PAP patients and GM-CSF knockout (KO) mice, suggesting PPARγ and ABCG1 involvement in surfactant catabolism. Because ABCG1 represents a PPARγ target, we hypothesized that PPARγ restoration would increase ABCG1 and reduce macrophage lipid accumulation. Upregulation of PPARγ was achieved using a lentivirus expression system in vivo. GM-CSF KO mice received intratracheal instillation of lentivirus (lenti)-PPARγ or control lenti-eGFP. Ten days postinstillation, 79% of harvested alveolar macrophages expressed eGFP, demonstrating transduction. Alveolar macrophages showed increased PPARγ and ABCG1 expression after lenti-PPARγ instillation, whereas PPARγ and ABCG1 levels remained unchanged in lenti-eGFP controls. Alveolar macrophages from lenti-PPARγ-treated mice also exhibited reduced intracellular phospholipids and increased cholesterol efflux to HDL, an ABCG1-mediated pathway. In vivo instillation of lenti-PPARγ results in: 1) upregulating ABCG1 and PPARγ expression of GM-CSF KO alveolar macrophages, 2) reducing intracellular lipid accumulation, and 3) increasing cholesterol efflux activity.

Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice.

We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPARγ) and the PPARγ-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPARγ. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPARγ plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p=0.0005) increase in ABCG1 expression with no change of PPARγ or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by analysis of bronchoalveolar lavage fluid. Lung compliance was diminished in untreated GMCSF KO mice but improved significantly after lenti-ABCG1 treatment. Data demonstrate that in vivo instillation of lenti-ABCG1 in GM-CSF KO mice is sufficient to restore pulmonary homeostasis by: (1) upregulating ABCG1; (2) reducing intra and extracellular lipids; and (3) improving lung function. Results suggest that the ABCG1 lipid transporter is the key downstream target of GM-CSF-induced PPARγ necessary for surfactant catabolism.

Deletion of PPAR gamma in alveolar macrophages is associated with a Th-1 pulmonary inflammatory response.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is constitutively expressed at high levels in healthy alveolar macrophages, in contrast to other tissue macrophages and blood monocytes. PPARgamma ligands have been shown to down-regulate IFN-gamma-stimulated inducible NO synthase (iNOS) in macrophages. Because NO is an important inflammatory mediator in the lung, we hypothesized that deletion of alveolar macrophage PPARgamma in vivo would result in up-regulation of iNOS and other inflammatory mediators. The loss of PPARgamma in macrophages was achieved by crossing floxed (+/+) PPARgamma mice and a transgenic mouse containing the CRE recombinase gene under the control of the murine M lysozyme promoter (PPARgammaKO). Alveolar macrophages were harvested by bronchoalveolar lavage (BAL). Lymphocytes (CD8:CD4 ratio = 2.8) were increased in BAL of PPARgammaKO vs wild-type C57BL6; p < or = 0.0001. Both iNOS and IFN-gamma expression were significantly elevated (p < or = 0.05) in BAL cells. Th-1 associated cytokines including IL-12 (p40), MIP-1alpha (CCL3), and IFN inducible protein-10 (IP-10, CXCL10) were also elevated. IL-4 and IL-17A were not detected. To test whether these alterations were due to the lack of PPARgamma, PPARgamma KO mice were intratracheally inoculated with a PPARgamma lentivirus construct. PPARgamma transduction resulted in significantly decreased iNOS and IFN-gamma mRNA expression, as well as reduced BAL lymphocytes. These results suggest that lack of PPARgamma in alveolar macrophages disrupts lung homeostasis and results in a Th1-like inflammatory response.

Targeted PPAR{gamma} deficiency in alveolar macrophages disrupts surfactant catabolism.

Surfactant accumulates in alveolar macrophages of granulocyte-macrophage colony-stimulating factor (GM-CSF) knockout (KO) mice and pulmonary alveolar proteinosis (PAP) patients with a functional loss of GM-CSF resulting from neutralizing anti-GM-CSF antibody. Alveolar macrophages from PAP patients and GM-CSF KO mice are de-ficient in peroxisome proliferator-activated receptor-gamma (PPARgamma) and ATP-binding cassette (ABC) lipid transporter ABCG1. Previous studies have demonstrated that GM-CSF induces PPARgamma. We therefore hypothesized that PPARgamma promotes surfactant catabolism through regulation of ABCG1. To address this hypothesis, macrophage-specific PPARgamma (MacPPARgamma) knockout mice were utilized. MacPPARgamma KO mice develop foamy, lipid-engorged Oil Red O positive alveolar macrophages. Lipid analyses revealed significant increases in the cholesterol and phospholipid contents of MacPPARgamma KO alveolar macrophages and extracellular bronchoalveolar lavage (BAL)-derived fluids. MacPPARgamma KO alveolar macrophages showed decreased expression of ABCG1 and a deficiency in ABCG1-mediated cholesterol efflux to HDL. Lipid metabolism may also be regulated by liver X receptor (LXR)-ABCA1 pathways. Interestingly, ABCA1 and LXRbeta expression were elevated, indicating that this pathway is not sufficient to prevent surfactant accumulation in alveolar macrophages. These results suggest that PPARgamma mediates a critical role in surfactant homeostasis through the regulation of ABCG1.

PPARgamma regulates the expression of cholesterol metabolism genes in alveolar macrophages.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear transcription factor involved in lipid metabolism that is constitutively expressed in the alveolar macrophages of healthy individuals. PPARgamma has recently been implicated in the catabolism of surfactant by alveolar macrophages, specifically the cholesterol component of surfactant while the mechanism remains unclear. Studies from other tissue macrophages have shown that PPARgamma regulates cholesterol influx, efflux, and metabolism. PPARgamma promotes cholesterol efflux through the liver X receptor-alpha (LXRalpha) and ATP-binding cassette G1 (ABCG1). We have recently shown that macrophage-specific PPARgamma knockout (PPARgamma KO) mice accumulate cholesterol-laden alveolar macrophages that exhibit decreased expression of LXRalpha and ABCG1 and reduced cholesterol efflux. We hypothesized that in addition to the dysregulation of these cholesterol efflux genes, the expression of genes involved in cholesterol synthesis and influx was also dysregulated and that replacement of PPARgamma would restore regulation of these genes. To investigate this hypothesis, we have utilized a Lentivirus expression system (Lenti-PPARgamma) to restore PPARgamma expression in the alveolar macrophages of PPARgamma KO mice. Our results show that the alveolar macrophages of PPARgamma KO mice have decreased expression of key cholesterol synthesis genes and increased expression of cholesterol receptors CD36 and scavenger receptor A-I (SRA-I). The replacement of PPARgamma (1) induced transcription of LXRalpha and ABCG1; (2) corrected suppressed expression of cholesterol synthesis genes; and (3) enhanced the expression of scavenger receptors CD36. These results suggest that PPARgamma regulates cholesterol metabolism in alveolar macrophages.

PPARγ Deficiency in Carbon Nanotube-elicited Granulomatous Inflammation Promotes a Th17 Response to a Microbial Antigen.

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.