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.

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.

Matrix Metalloproteinase-12 Is Required for Granuloma Progression.

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.

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.

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.

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.

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.

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.

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.

Exposure to a Mycobacterial Antigen, ESAT-6, Exacerbates Granulomatous and Fibrotic Changes in a Multiwall Carbon Nanotube Model of Chronic Pulmonary Disease.

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.

Carbon Nanotubes and Chronic Granulomatous Disease.

Use of nanomaterials in manufactured consumer products is a rapidly expanding industry and potential toxicities are just beginning to be explored. Combustion-generated multiwall carbon nanotubes (MWCNT) or nanoparticles are ubiquitous in non-manufacturing environments and detectable in vapors from diesel fuel, methane, propane, and natural gas. In experimental animal models, carbon nanotubes have been shown to induce granulomas or other inflammatory changes. Evidence suggesting potential involvement of carbon nanomaterials in human granulomatous disease, has been gathered from analyses of dusts generated in the World Trade Center disaster combined with epidemiological data showing a subsequent increase in granulomatous disease of first responders. In this review we will discuss evidence for similarities in the pathophysiology of carbon nanotube-induced pulmonary disease in experimental animals with that of the human granulomatous disease, sarcoidosis.

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.

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.

Alveolar macrophage cathelicidin deficiency in severe sarcoidosis.

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.

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.

Alveolar macrophage activation in obese patients with obstructive sleep apnea.

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.