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

Abstract

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.

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

“…Our previous studies noted that MWCNT-mediated granulomatous inflammation was exacerbated in mice with macrophage-specific PPARγ deficiency [22], therefore it was of interest to determine whether ESAT-6 effects were associated with reduced PPARγ expression. Results indicated that addition of ESAT-6 to MWCNT did not further reduce PPARγ mRNA expression in BAL cells (Figure 6).…”

“…C57BL/6J wild-type mice received an oropharyngeal instillation of MWCNT after sedation with isofluorane. MWCNTs (catalogue number 900–1501, lot GS1801, SES Research, Houston, TX) were freshly prepared and have been extensively described previously [10,22]. A single pulmonary instillation of MWCNT (100 μg) in PBS/35% surfactant (vehicle) ± ESAT-6 peptide 14 (NNALQNLARTISEAG) (20 μg) were delivered to wild-type C57Bl/6 mice.…”

“…Sham controls received vehicle alone; additional controls received only ESAT-6. Animals were sacrificed at 60 days post instillation for evaluation of lung tissue histopathology, laser capture microdissection (LCM) of granulomas, and bronchoalveolar lavage (BAL) cells as previously described [22]. …”

“…A semiquantitative scoring system previously described [22] was used for a relative comparison of the numbers and quality of the granulomas formed in MWCNT versus ESAT-6 + MWCNT instilled mice. Hematoxylin and Eosin stained sections of lung from each of the experimental mice was assigned a score of between 0 and 5 by two independent investigators using the following scoring system: (score 0) – no granulomas or aggregates of macrophages seen; (score 1) - few small groups of macrophages but no well-formed granulomas; (score 2) - scattered small granulomas not easily seen on scanning power (20×); (score 3) - scattered small granulomas easily seen on scanning power (20×); (score 4) - scattered small granulomas with occasional larger granulomas seen on scanning power (20×); (score 5) - numerous large granulomas easily seen on scanning power.…”

Etiologies of Sarcoidosis

“…HP has been reported in metalworkers that were exposed to nontuberculous mycobacteria in contaminated metalworking fluid, providing an interesting example of a different pathogenic outcome from exposure to these organisms that results in a form of granulomatous inflammation in the lung [64]. Recent studies demonstrate that carbon nanotubes can induce some features of a sarcoidosis-like granulomatous reaction when injected into the lungs of mice, but fail to recapitulate the clinical features of sarcoidosis [65][66][67]. Whether the definition of sarcoidosis should include granulomatous responses in the lung induced by exposure to such known inorganic or organic agents remains a contested issue [68].…”