Carbon nanotube-induced pulmonary granulomatous disease: Twist1 and alveolar macrophage M1 activation

Abstract

Sarcoidosis, a chronic granulomatous disease of unknown cause, has been linked to several environmental risk factors, among which are some that may favor carbon nanotube formation. Using gene array data, we initially observed that bronchoalveolar lavage (BAL) cells from sarcoidosis patients displayed elevated mRNA of the transcription factor, Twist1, among many M1-associated genes compared to healthy controls. Based on this observation we hypothesized that Twist1 mRNA and protein expression might become elevated in alveolar macrophages from animals bearing granulomas induced by carbon nanotube instillation. To address this hypothesis, wild-type and macrophage-specific peroxisome proliferator-activated receptor gamma (PPARγ) knock out mice were given oropharyngeal instillation of multiwall carbon nanotubes (MWCNT). BAL cells obtained 60 days later exhibited significantly elevated Twist1 mRNA expression in granuloma-bearing wild-type or PPARγ knock out alveolar macrophages compared to sham controls. Overall, Twist1 expression levels in PPARγ knock out mice were higher than those of wild-type. Concurrently, BAL cells obtained from sarcoidosis patients and healthy controls validated gene array data: qPCR and protein analysis showed significantly elevated Twist1 in sarcoidosis compared to healthy controls. In vitro studies of alveolar macrophages from healthy controls indicated that Twist1 was inducible by classical (M1) macrophage activation stimuli (LPS, TNFα) but not by IL-4, an inducer of alternative (M2) macrophage activation. Findings suggest that Twist1 represents a PPARγ-sensitive alveolar macrophage M1 biomarker which is induced by inflammatory granulomatous disease in the MWCNT model and in human sarcoidosis.

Figure 1.

(A–C). Twist1 in human alveolar macrophages. Twist1 mRNA expression is intrinsically elevated in alveolar macrophages of sarcoidosis patients compared to healthy controls (A); Immunostaining did not detect Twist1 protein expression in healthy control alveolar macrophages (B) but Twist1 protein appears in alveolar macrophages of sarcoidosis patients; scale bar = 50 μm (C). Cells were counterstained with propidium iodide to highlight nuclei (same magnification as Figure 1B). Images are representative of findings from three patients and three controls.

Figure 1.

(A–C). Twist1 in human alveolar macrophages. Twist1 mRNA expression is intrinsically elevated in alveolar macrophages of sarcoidosis patients compared to healthy controls (A); Immunostaining did not detect Twist1 protein expression in healthy control alveolar macrophages (B) but Twist1 protein appears in alveolar macrophages of sarcoidosis patients; scale bar = 50 μm (C). Cells were counterstained with propidium iodide to highlight nuclei (same magnification as Figure 1B). Images are representative of findings from three patients and three controls.

Figure 2.

(A–C). Upregulation of Twist1 mRNA is found in healthy control alveolar macrophages exposed in vitro to M1 activators, LPS (A); PAM3-CSK4 (B); and TNFα (C).

Figure 2.

(A–C). Upregulation of Twist1 mRNA is found in healthy control alveolar macrophages exposed in vitro to M1 activators, LPS (A); PAM3-CSK4 (B); and TNFα (C).

Figure 3.

Twist1 in murine alveolar macrophages. Bronchoalveolar lavage (BAL) cells were obtained from C57Bl/6 wild-type and macrophage specific PPARγ KO mice at 60 days after oropharyngeal instillation of MWCNT. At this time point, all animals have extensive granulomatous disease in the lung [7,20]. Twist1 mRNA is elevated in BAL cells from MWCNT-treated wild-type (n = 7) mice versus sham controls (n = 6) as well as in MWCNT-treated macrophage specific PPARγ KO mice (n = 11) compared to sham controls (n = 7) (A); Twist1 mRNA is intrinsically elevated in BAL from untreated PPARγ KO mice compared to untreated wild-type controls (B); Immunostaining of alveolar macrophages from wild-type C57Bl/6 mice does not detect Twist1 protein after sham-treatment. Scale bar = 5 μm. (C); but shows elevated Twist1 protein after MWCNT instillation (same magnification as Figure 3C) (D).