Expression of microRNA in Alveolar Macrophages Deficient in PPARy

The nuclear transcription factor Peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of macrophage activation and inflammatory mediators. Alveolar macrophages of healthy individuals constitutively express PPARgamma Decreased activity and expression of PPARgamma are observed in the alveolar macrophages from patients suffering from inflammatory conditions such as pulmonary alveolar proteinosis (PAP) and sarcoidosis. These finding suggest that PPARf× activity may have an integral role in maintaining lung homeostasis. This study tested the hypothesis that microRNA expression would be dysregulated in murine alveolar macrophages deficient in PPARgamma. microRNA (miR) are small non-coding RNA molecules that post-transcriptionally regulate the expression of messenger RNA.   Evaluation of microRNA in the murine model of PAP, the GM-CSF-KO mouse, demonstrates the elevation of miR-27a and miR-27b which target PPARgamma. The deficiency of PPARgamma and the lipid transporters ABCA1 and ABCG1 have been shown to contribute to the pathology of PAP. The microRNA miR-33-3p and miR-33-5p, which target these lipid transporters, were also elevated in GM-CSF-KO mice. Pulmonary granulomas comparable to those observed in pulmonary sarcoidosis are induced by instillation of multiwall carbon nanotubes (MWCNT) in C57Bl/6 mice. These animals have decreased PPARgamma activity and show elevated expression of miR-27a and miR-27b. It was also observed that the expression of the transporters ABCA1 and ABCG1 were decreased in MWCNT instilled mice. Expression of miR-33-3p and miR-33-5p was elevated in MWCNT instilled animals. The expression of microRNA that affects the activity of NF-£eB is also elevated in both murine models.  We next investigated the use of PPARgamma agonist rosiglitazone on the expression of microRNA and messenger RNA. The use of rosiglitazone altered the expression of microRNA in both GM-CSF-KO and C57Bl/6+MWCNT mice. Rosiglitazone treatment altered the expression of the lipid transporter ABCA1and ABCG1 in C57Bl/6+MWCNT mice. The elevation of proinflammatory cytokines was also observed.   Taken together, these observations support the hypothesis that PPARgamma activity effects the microRNA and gene expression in alveolar macrophages which is critical to overall lung homeostasis. Understanding the relationship between PPARgamma and microRNA in alveolar macrophage biology will provide insight into the regulation of the lung environment and possible therapeutic targets.  M.S

THE ROLE OF THE ALVEOLAR MACROPHAGE IN CARBON NANOTUBE ELICITED MURINE MODEL OF PULMONARY GRANULOMATOUS INFLAMMATION

Pulmonary sarcoidosis is a debilitating inflammatory condition characterized by the presence of granulomatous lesions throughout the lung. Granulomas are a physiological response to inhaled antigens or particulate matter which cannot be properly degraded. To explore mechanisms of granuloma formation and maintenance our laboratory developed a murine model of pulmonary granulomatous inflammation elicited by multi-wall carbon nanotubes (MWCNT). We have found the MWCNT model bears striking similarities to pulmonary sarcoidosis pathophysiology, including increased expression of inflammatory mediators and decreased expression and activity of peroxisome proliferator activated receptor-gamma (PPARy) in alveolar macrophages. PPARy is a known regulator of macrophage activation and serves a crucial role in pulmonary lipid homeostasis through the regulation of macrophage ATP-binding cassette (ABC) lipid transporter-G1 (ABCG1). Further studies demonstrated that alveolar macrophages obtained from sarcoidosis patients and MWCNT instilled animals have decreased gene expression and protein levels of ABCG1 and ABCA1, a complementary cholesterol transporter. These studies aim to further define the role of alveolar macrophage PPARy, ABCA1 and ABCG1 in pulmonary granulomatous inflammation. We hypothesized that deficiency of ABCA1 and ABCG1 would exacerbate MWCNT induced granuloma formation. To test this hypothesis, macrophage-specific ABCA1, ABCG1 or combined ABCA1/ABCG1 knockout mice were developed and evaluated following MWCNT instillation. We found that deficiency of ABCG1 but not ABCA1 leads to a significant upregulation of pro-inflammatory mediators and promotes pulmonary granuloma formation. Interestingly, the combined deficiency of ABCA1/ABCG1 leads to an exacerbated pulmonary inflammatory phenotype. We further hypothesized that upregulation of the PPARy-ABCG1 pathway would limit MWCNT induced granuloma formation and inflammation. To test this hypothesis, we administered the PPARy-specific ligand rosiglitazone to MWCNT instilled animals and evaluated the effect on granulomatous inflammation. We found that the administration of rosiglitazone promotes the expression of alveolar macrophage ABCG1, limits the severity of MWCNT induced granuloma formation and reduced alveolar macrophage pro-inflammatory gene expression. These studies suggest that the PPAR-ABCG1 pathway, specifically the deficiency of alveolar macrophage ABCG1 plays a significant role in pulmonary granulomatous inflammation

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.ECU Open Access Publishing Support Fun

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

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

High-resolution genetic mapping with pooled sequencing

Background: Modern genetics has been transformed by high-throughput sequencing. New experimental designs in model organisms involve analyzing many individuals, pooled and sequenced in groups for increased efficiency. However, the uncertainty from pooling and the challenge of noisy sequencing data demand advanced computational methods. Results: We present MULTIPOOL, a computational method for genetic mapping in model organism crosses that are analyzed by pooled genotyping. Unlike other methods for the analysis of pooled sequence data, we simultaneously consider information from all linked chromosomal markers when estimating the location of a causal variant. Our use of informative sequencing reads is formulated as a discrete dynamic Bayesian network, which we extend with a continuous approximation that allows for rapid inference without a dependence on the pool size. MULTIPOOL generalizes to include biological replicates and case-only or case-control designs for binary and quantitative traits. Conclusions: Our increased information sharing and principled inclusion of relevant error sources improve resolution and accuracy when compared to existing methods, localizing associations to single genes in several cases. MULTIPOOL is freely available at http://cgs.csail.mit.edu/multipool/ webcite.National Science Foundation (U.S.) (Graduate Research Fellowship Grant 0645960

Transcriptomic epidemiology of smoking: the effect of smoking on gene expression in lymphocytes

<p>Abstract</p> <p>Background</p> <p>This investigation offers insights into system-wide pathological processes induced in response to cigarette smoke exposure by determining its influences at the gene expression level.</p> <p>Methods</p> <p>We obtained genome-wide quantitative transcriptional profiles from 1,240 individuals from the San Antonio Family Heart Study, including 297 current smokers. Using lymphocyte samples, we identified 20,413 transcripts with significantly detectable expression levels, including both known and predicted genes. Correlation between smoking and gene expression levels was determined using a regression model that allows for residual genetic effects.</p> <p>Results</p> <p>With a conservative false-discovery rate of 5% we identified 323 unique genes (342 transcripts) whose expression levels were significantly correlated with smoking behavior. These genes showed significant over-representation within a range of functional categories that correspond well with known smoking-related pathologies, including immune response, cell death, cancer, natural killer cell signaling and xenobiotic metabolism.</p> <p>Conclusions</p> <p>Our results indicate that not only individual genes but entire networks of gene interaction are influenced by cigarette smoking. This is the largest <it>in vivo </it>transcriptomic epidemiological study of smoking to date and reveals the significant and comprehensive influence of cigarette smoke, as an environmental variable, on the expression of genes. The central importance of this manuscript is to provide a summary of the relationships between gene expression and smoking in this exceptionally large cross-sectional data set.</p

An attainable global vision for conservation and human well-being

A hopeful vision of the future is a world in which both people and nature thrive, but there is little evidence to support the feasibility of such a vision. We used a global, spatially explicit, systems modeling approach to explore the possibility of meeting the demands of increased populations and economic growth in 2050 while simultaneously advancing multiple conservation goals. Our results demonstrate that if, instead of “business as usual” practices, the world changes how and where food and energy are produced, this could help to meet projected increases in food (54%) and energy (56%) demand while achieving habitat protection (>50% of natural habitat remains unconverted in most biomes globally; 17% area of each ecoregion protected in each country), reducing atmospheric greenhouse-gas emissions consistent with the Paris Climate Agreement (≤1.6°C warming by 2100), ending overfishing, and reducing water stress and particulate air pollution. Achieving this hopeful vision for people and nature is attainable with existing technology and consumption patterns. However, success will require major shifts in production methods and an ability to overcome substantial economic, social, and political challenges

Expression of microRNA in Alveolar Macrophages Deficient in PPARy

The nuclear transcription factor Peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of macrophage activation and inflammatory mediators. Alveolar macrophages of healthy individuals constitutively express PPARgamma Decreased activity and expression of PPARgamma are observed in the alveolar macrophages from patients suffering from inflammatory conditions such as pulmonary alveolar proteinosis (PAP) and sarcoidosis. These finding suggest that PPARf× activity may have an integral role in maintaining lung homeostasis. This study tested the hypothesis that microRNA expression would be dysregulated in murine alveolar macrophages deficient in PPARgamma. microRNA (miR) are small non-coding RNA molecules that post-transcriptionally regulate the expression of messenger RNA. Evaluation of microRNA in the murine model of PAP, the GM-CSF-KO mouse, demonstrates the elevation of miR-27a and miR-27b which target PPARgamma. The deficiency of PPARgamma and the lipid transporters ABCA1 and ABCG1 have been shown to contribute to the pathology of PAP. The microRNA miR-33-3p and miR-33-5p, which target these lipid transporters, were also elevated in GM-CSF-KO mice. Pulmonary granulomas comparable to those observed in pulmonary sarcoidosis are induced by instillation of multiwall carbon nanotubes (MWCNT) in C57Bl/6 mice. These animals have decreased PPARgamma activity and show elevated expression of miR-27a and miR-27b. It was also observed that the expression of the transporters ABCA1 and ABCG1 were decreased in MWCNT instilled mice. Expression of miR-33-3p and miR-33-5p was elevated in MWCNT instilled animals. The expression of microRNA that affects the activity of NF-£eB is also elevated in both murine models. We next investigated the use of PPARgamma agonist rosiglitazone on the expression of microRNA and messenger RNA. The use of rosiglitazone altered the expression of microRNA in both GM-CSF-KO and C57Bl/6+MWCNT mice. Rosiglitazone treatment altered the expression of the lipid transporter ABCA1and ABCG1 in C57Bl/6+MWCNT mice. The elevation of proinflammatory cytokines was also observed. Taken together, these observations support the hypothesis that PPARgamma activity effects the microRNA and gene expression in alveolar macrophages which is critical to overall lung homeostasis. Understanding the relationship between PPARgamma and microRNA in alveolar macrophage biology will provide insight into the regulation of the lung environment and possible therapeutic targets

THE ROLE OF THE ALVEOLAR MACROPHAGE IN CARBON NANOTUBE ELICITED MURINE MODEL OF PULMONARY GRANULOMATOUS INFLAMMATION

Pulmonary sarcoidosis is a debilitating inflammatory condition characterized by the presence of granulomatous lesions throughout the lung. Granulomas are a physiological response to inhaled antigens or particulate matter which cannot be properly degraded. To explore mechanisms of granuloma formation and maintenance our laboratory developed a murine model of pulmonary granulomatous inflammation elicited by multi-wall carbon nanotubes (MWCNT). We have found the MWCNT model bears striking similarities to pulmonary sarcoidosis pathophysiology , including increased expression of inflammatory mediators and decreased expression and activity of peroxisome proliferator activated receptor-gamma (PPARy) in alveolar macrophages. PPARy is a known regulator of macrophage activation and serves a crucial role in pulmonary lipid homeostasis through the regulation of macrophage ATP-binding cassette (ABC) lipid transporter-G1 (ABCG1). Further studies demonstrated that alveolar macrophages obtained from sarcoidosis patients and MWCNT instilled animals have decreased gene expression and protein levels of ABCG1 and ABCA1 , a complementary cholesterol transporter. These studies aim to further define the role of alveolar macrophage PPARy , ABCA1 and ABCG1 in pulmonary granulomatous inflammation. We hypothesized that deficiency of ABCA1 and ABCG1 would exacerbate MWCNT induced granuloma formation. To test this hypothesis , macrophage-specific ABCA1 , ABCG1 or combined ABCA1/ABCG1 knockout mice were developed and evaluated following MWCNT instillation. We found that deficiency of ABCG1 but not ABCA1 leads to a significant upregulation of pro-inflammatory mediators and promotes pulmonary granuloma formation. Interestingly , the combined deficiency of ABCA1/ABCG1 leads to an exacerbated pulmonary inflammatory phenotype. We further hypothesized that upregulation of the PPARy-ABCG1 pathway would limit MWCNT induced granuloma formation and inflammation. To test this hypothesis , we administered the PPARy-specific ligand rosiglitazone to MWCNT instilled animals and evaluated the effect on granulomatous inflammation. We found that the administration of rosiglitazone promotes the expression of alveolar macrophage ABCG1 , limits the severity of MWCNT induced granuloma formation and reduced alveolar macrophage pro-inflammatory gene expression. These studies suggest that the PPAR-ABCG1 pathway , specifically the deficiency of alveolar macrophage ABCG1 plays a significant role in pulmonary granulomatous inflammation