Novel Murine Model of Chronic Granulomatous Lung Inflammation Elicited by Carbon Nanotubes

Abstract

Lung granulomas are associated with numerous conditions, including inflammatory disorders, exposure to environmental pollutants, and infection. Osteopontin is a chemotactic cytokine produced by macrophages, and is implicated in extracellular matrix remodeling. Furthermore, osteopontin is up-regulated in granulomatous disease, and osteopontin null mice exhibit reduced granuloma formation. Animal models currently used to investigate chronic lung granulomatous inflammation bear a pathological resemblance, but lack the chronic nature of human granulomatous disease. Carbon nanoparticles are generated as byproducts of combustion. Interestingly, experimental exposures to carbon nanoparticles induce pulmonary granuloma-like lesions. However, the recruited cellular populations and extracellular matrix gene expression profiles within these lesions have not been explored. Because of the rapid resolution of granulomas in current animal models, the mechanisms responsible for persistence have been elusive. To overcome the limitations of previous models, we investigated whether a model using multiwall carbon nanoparticles would resemble chronic human lung granulomatous inflammation. We hypothesized that pulmonary exposure to multiwall carbon nanoparticles would induce granulomas, elicit a macrophage and T-cell response, and mimic other granulomatous disorders with an up-regulation of osteopontin. This model demonstrates: (1) granulomatous inflammation, with macrophage and T-cell infiltration; (2) resemblance to the chronicity of human granulomas, with persistence up to 90 days; and (3) a marked elevation of osteopontin, metalloproteinases, and cell adhesion molecules in granulomatous foci isolated by laser-capture microdissection and in alveolar macrophages from bronchoalveolar lavage. The establishment of such a model provides an important platform for mechanistic studies on the persistence of granuloma.

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

“…Our previous MWCNT studies utilized trichrome staining but detected no fibrosis after instillation of MWCNT alone [10]. Trichrome staining in the current study revealed marked fibrotic changes within granulomas of mice receiving MWCNT + ESAT 6 (Figure 2D) as compared to granulomas of mice receiving only MWCNT (Figure 2C).…”

“…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.…”

“…Leukocyte differential counts of BAL cells were calculated from cytospins (100 cells/40 × high power field × 3 fields) as previously described [10]. Aliquots of BAL cells were centrifuged and frozen for qPCR evaluation as previously described [23].…”

“…Primer-probe sets for CCL2 (MCP1), CCL4 (MIP1β), Fibronectin 1 (Fn1), IFN-γ, PPARγ, Matrix Metalloproteinase-12 (MMP12), osteopontin (OPN), CD3e, and the GAPDH housekeeping gene were obtained from Qiagen, Germantown, MD. Data were expressed as fold change in mRNA expression compared to control values as previously described [10]. Levels of OPN protein in BAL fluid were determined by ELISA assay (R&D Systems, Inc., Minneapolis, MN).…”

“…In order to explore pathophysiologic mechanisms of granuloma formation and persistence, we developed a novel multiwall carbon nanotube (MWCNT) model of granulomatous disease [10]. MWCNT-elicited granulomatous disease is chronic (where granulomas persist up to 90 days), and is characterized by persistently elevated pro-inflammatory cytokines together with T cell and macrophage recruitment – all traits found in sarcoidosis, a human granulomatous disease of unknown etiology [11].…”