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

ABSTRACT: 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.

SUBMITTER: Mohan A

PROVIDER: S-EPMC5966779 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: A well-validated animal model of sarcoidosis can help advance our understanding of the pathobiology of this complex disease. We have developed a multiwall carbon nanotube (MWCNT) based murine model that shows marked histological and inflammatory signal similarities to human sarcoidosis. In this study, we compared the alveolar immune cell transcriptional signatures of our murine model with human sarcoidosis to comprehensively assess overlapping molecular programs. Methods: Total RNA was extracted from alveolar macrophages of 6 MWCNT mice and 6 control mice and hybridized to Illumina Mouse WG-6 v2 microarrays. Transcriptional profiling of alveolar immune cells in 15 sarcoidosis patients and 12 healthy humans was previously performed using Affymetrix Human GeneChip U133A 2.0 microarrays. Rigorous statistical methods were used to identify differentially expressed genes. To better elucidate gene expression pathways, integrated network and gene set enrichment analysis (GSEA) were applied. To confirm differential expression of select network genes, we performed qPCR studies using the ABI Prism 7300 Detection. Results: We identified over 1000 genes that were differentially expressed between the control and the MWCNT mice. Gene Ontology functional analysis showed over-representation of processes primarily involved in immunity and inflammation in MCWNT mice. Applying a more comprehensive pathway analysis using 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 selective gene set enrichment between MWCNT mice and sarcoidosis patients were also observed, among them IL-27 signaling, NK-mediated cytotoxicity and proteasome pathways. We applied network analysis to differentially expressed genes common between the MWCNT model and human sarcoidosis to identify key drivers of disease and putative therapeutic targets. Conclusions: Our MWCNT murine model has an alveolar macrophage signature that varies significantly from control mice. Application of an unbiased transcriptomic approach revealed substantial functional similarities between a murine MWCNT model and human sarcoidosis particularly with respect to activation of immune-specific pathways.

Project description:RationaleThe critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis.ObjectivesTo determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis.MethodsSerum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-kappaB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase-peroxidase.Measurements and main resultsWe found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3(+) lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-kappaB in Toll-like receptor-2-expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-gamma, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2.ConclusionsSerum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.

Project description:Human exposure to carbon nanotubes (CNT) has been associated with the development of pulmonary sarcoid-like granulomatous disease. Our previous studies demonstrated that multi-walled carbon nanotubes (MWCNT) induced chronic pulmonary granulomatous inflammation in mice. Granuloma formation was accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) and disrupted intracellular lipid homeostasis in alveolar macrophages. Others have shown that PPARγ activation increases mitochondrial fatty acid oxidation (FAO) to reduce free fatty acid accumulation. Hence, we hypothesized that the disrupted lipid metabolism suppresses mitochondrial FAO. To test our hypothesis, C57BL/6 J mice were instilled by an oropharyngeal route with 100 μg MWCNT freshly suspended in 35 % Infasurf. Control sham mice received vehicle alone. Sixty days following instillation, mitochondrial FAO was measured in permeabilized bronchoalveolar lavage (BAL) cells. MWCNT instillation reduced the mitochondrial oxygen consumption rate of BAL cells in the presence of palmitoyl-carnitine as mitochondrial fuel. MWCNT also reduced mRNA expression of mitochondrial genes regulating FAO, carnitine palmitoyl transferase-1 (CPT1), carnitine palmitoyl transferase-2 (CPT2), hydroxyacyl-CoA dehydrogenase subunit beta (HADHB), and PPARγ coactivator 1 alpha (PPARGC1A). Importantly, both oxidative stress and apoptosis in alveolar macrophages and lung tissues of MWCNT-instilled mice were increased. Because macrophage PPARγ expression has been reported to be controlled by miR-27b which is known to induce oxidative stress and apoptosis, we measured the expression of miR-27b. Results indicated elevated levels in alveolar macrophages from MWCNT-instilled mice compared to controls. Given that inhibition of FAO and apoptosis are linked to M1 and M2 macrophage activation, respectively, the expression of both M1 and M2 key indicator genes were measured. Interestingly, results showed that both M1 and M2 phenotypes of alveolar macrophages were activated in MWCNT-instilled mice. In conclusion, alveolar macrophages of MWCNT-instilled mice had increased miR-27b expression, which may reduce the expression of PPARγ resulting in attenuation of FAO. This reduction in FAO may lead to activation of M1 macrophages. The upregulation of miR-27b may also induce apoptosis, which in turn can cause M2 activation of alveolar macrophages. These observations indicate a possible role of miR-27b in impaired mitochondrial function in the chronic activation of alveolar macrophages by MWCNT and the development of chronic pulmonary granulomatous inflammation.

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

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