BackgroundAlthough 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.MethodsWild-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.ResultsIn 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.ConclusionsOverall, data indicate that PPARγ deficiency promotes inflammation and granuloma formation, suggesting that PPARγ functions as a negative regulator of chronic granulomatous inflammation.

Quantification of 3D morphology and measurement of cellular functions were performed on the mouse melanoma cell lines of B16F10 to investigate the intriguing problem of structure-function relations in the genetically engineered cells with GPR4 overexpression. Results of 3D analysis of cells in suspension and phase contrast imaging of adherent cells yield consistent evidence that stimulation of the proton-sensing GPR4 receptor in these cells may modify significantly their morphology with diminishing ability to produce membrane protrusions and to migrate. Examination of the 3D parameters of mitochondria provide further insights on the measured variation of the maximal capacity of oxygen consumption rate among the genetically modified cells, indicating that the proton-sensing receptor may regulate cancer cell metabolism with increased mitochondrial surface area. Our study demonstrates clearly the significant benefits of quantitative 3D morphological study in illuminating cellular functions and development of novel morphology based cell assay methods.

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.

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