PPAR-gamma pathways attenuate pulmonary granuloma formation in a carbon nanotube induced murine model of sarcoidosis

Abstract

Peroxisome proliferator activated receptor gamma (PPARγ), a ligand activated nuclear transcription factor, is constitutively expressed in alveolar macrophages of healthy individuals. PPARγ deficiencies have been noted in several lung diseases including the alveolar macrophages of pulmonary sarcoidosis patients. We have previously described a murine model of multiwall carbon nanotubes (MWCNT) induced pulmonary granulomatous inflammation which bears striking similarities to pulmonary sarcoidosis, including the deficiency of alveolar macrophage PPARγ. Further studies demonstrate alveolar macrophage PPARγ deficiency exacerbates MWCNT-induced pulmonary granulomas. Based on these observations we hypothesized that activation of PPARγ via administration of the PPARγ-specific ligand rosiglitazone would limit MWCNT-induced granuloma formation and promote PPARγ-dependent pathways. Results presented here show that rosiglitazone significantly limits the frequency and severity of MWCNT-induced pulmonary granulomas. Furthermore, rosiglitazone attenuates alveolar macrophage NF-κB activity and downregulates the expression of the pro-inflammatory mediators, CCL2 and osteopontin. PPARγ activation via rosiglitazone also prevents the MWCNT-induced deficiency of PPARγ-regulated ATP-binding cassette lipid transporter-G1 (ABCG1) expression. ABCG1 is crucial to pulmonary lipid homeostasis. ABCG1 deficiency results in lipid accumulation which promotes pro-inflammatory macrophage activation. Our results indicate that restoration of homeostatic ABCG1 levels by rosiglitazone correlates with both reduced pulmonary lipid accumulation, and decreased alveolar macrophage activation. These data confirm and further support our previous observations that PPARγ pathways are critical in regulating MWCNT-induced pulmonary granulomatous inflammation.

Keywords: Alveolar macrophage; Carbon nanotube; Granuloma; Inflammation; Lipid transporters; Sarcoidosis.

Fig. 1.. Rosiglitazone Regulates Alveolar Macrophage Gene Expression 10 days following MWCNT instillation.

Quantitative-PCR analysis of the lipid regulatory genes (A) peroxisome proliferator activated receptor-γ (PPARγ), ATP-binding cassette lipid transporter-G1 (ABCG1), and ABCA1; and inflammatory mediators (B) CCL2 and osteopontin (OPN) in BAL cells of vehicle or MWCNT-instilled mice. Effect of rosiglitazone dose curve on alveolar macrophage (C) ABCG1, (D) CCL2 and (E) osteopontin expression. ★p < 0.05, n ≥ 3/ treatment.

Fig. 2.. Rosiglitazone Inhibits Pulmonary Granuloma Formation 20 days post MWCNT Instillation.

Quantitative analysis of H&E stained lung tissue was utilized to evaluate (A) the average number of granulomas, (B) average granuloma size, (C) and total carbon deposition in MWCNT-instilled animals receiving normal chow or rosiglitazone (6 mg/kg). Evaluation of (D) mediastinal lymph node volume and (E) BAL-fluid protein concentration in vehicle and MWCNT-instilled animals receiving normal chow or rosiglitazone (6 mg/ kg). ★p < 0.05, n ≥ 5/treatment.

Fig. 3.. Rosiglitazone Reduces Alveolar Macrophage Activation 20 days post MWCNT-Instillation.

Quantitative-PCR analysis of (A) CCL2 and (B) osteopontin expression in BAL cells of vehicle or MWCNT-instilled animals receiving normal chow or rosiglitazone (6 mg/kg). (C) Mean fluorescence intensity of nuclear NF-κB p65 protein in MWCNT-instilled animals receiving normal chow or rosiglitazone (6 mg/kg), ≤99 cells/animal. ★p < 0.01, n ≥ 5/treatment.

Fig. 4.. Rosiglitazone limits pulmonary dyslipidemia 20 days following MWCNT-instillation.

Quantitative-PCR analysis of the lipid regulatory genes (A) PPARγ (B) ABCA1, (C) ABCG1 and (D) CD36 in vehicle or MWCNT-instilled animals receiving normal chow or rosiglitazone (6 mg/kg). Evaluation of (E) mean alveolar macrophage diameter and (E) total cholesterol content of BAL fluid of vehicle and MWCNT-instilled mice receiving normal chow or rosiglitazone (6 mg/kg). ★p < 0.01, n ≥ 5/treatment.