Elevated MicroRNA-33 in Sarcoidosis and a Carbon Nanotube Model of Chronic Granulomatous Disease

Abstract

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.

Keywords: carbon nanotube; lipid transporters; microRNA; murine model; sarcoidosis.

Figure 1.

Multiwall carbon nanotube (MWCNT) instillation dysregulates lipid metabolism and elevates microRNA (miR or miRNA)-33 expression in alveolar macrophages and granuloma tissues. (A). Quantitative PCR (qPCR) analysis of lipid transporter ATP-binding cassette (ABC) A1 and ABCG1 mRNA indicates repression in bronchoalveolar lavage (BAL) cells from wild-type mice after MWCNT instillation compared with sham-treated (PBS/surfactant) mice. (B) ABCA1 and ABCG1 proteins are reduced in MWCNT-treated wild-type mice compared with sham controls, as detected by capillary Western method. (C) Macrophage-specific peroxisome proliferator–activated receptor γ (PPARγ) knockout (KO) mice display repressed ABCA1 and ABCG1 mRNA at 60 days after MWCNT instillation, as assayed by qPCR. (D) Capillary Western analysis demonstrates decreased ABCA1/ABCG1 proteins in PPARγ KO mice. (E) Oil red O staining indicative of intracellular neutral lipid accumulation is increased in alveolar macrophages of wild-type mice after MWCNT instillation. (F) miR-33 is elevated in BAL cells from MWCNT-instilled mice and is also intrinsically elevated in BAL cells from untreated PPARγ KO (control) mice compared with untreated wild-type C57 controls. Laser capture microdissection was used to dissect granuloma [G(+)] from nongranuloma [G(−)] tissues for qPCR analysis. Significant elevation of miR-33 compared with sham controls was only found in granulomatous tissues from wild-type (G) or macrophage-specific PPARγ KO mice (H). Data presented are means ± SEM; * denotes P value and number of samples per group.

Figure 2.

miR-33 overexpression in vitro represses alveolar macrophage lipid transporters. Alveolar macrophages from wild-type mice were adhered and cultured in vitro for 48 hours with medium, lentivirus control (Lenti-control), or lentivirus–miR-33 (Lenti–miR-33; 25 ng) before qPCR analysis. Lenti–miR-33 elevated miR-33 expression (A) and repressed lipid transporter ABCA1 (B) and ABCG1 (C) expression compared with Lenti-controls. (D) PPARγ mRNA expression was unaffected by lenti–miR-33 overexpression. Data presented are means ± SEM; * denotes P value and number of samples per group.

Figure 3.

Lipid transporters ABCA1 and ABCG1 were repressed and miR-33 was elevated in patients with sarcoidosis. (A) Expression of both ABCA1 and ABCG1 mRNA was repressed in sarcoidosis BAL cells compared with healthy control cells, as analyzed by qPCR. (B) Capillary Western analysis demonstrates reduced ABCA1 and ABCG1 proteins in sarcoidosis BAL cells. (C) qPCR analysis showed elevated miR-33 in sarcoidosis BAL cells compared with healthy control cells. (D) Histopathology of control (cardiac patient) lymph node. (E) Histopathology of sarcoidosis lymph nodes showed extensive granulomatous changes. (F) qPCR analysis illustrates elevated miR-33 expression in sarcoidosis lymph nodes (n = 7) compared with control nodes (n = 4). Data presented are means ± SEM; * denotes P value and number of samples per group.

Figure 4.

Proposed mechanism of ABCA1 and ABCG1 involvement in chronic granulomatous disease. MWCNT uptake activates alveolar macrophages and elicits an inflammatory response of cytokines and chemokines. As a result, miR-33 is elevated and PPARγ is repressed, leading to decreased ABCA1 and ABCG1 lipid transporter expression, respectively, thus increasing intracellular cholesterol, which further exacerbates the inflammatory response.