Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model.

Eman Soliman,David Ogburn,Nancy Leffler,Achut G Malur,Ahmed E M Elhassanny,Anagha Malur,Sophia Bhalla,Mary Jane Thomassen

doi:10.3390/ijms23010047

Abstract

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-β) expression in lungs, together with an increased expression of TGF-β related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin β3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.

Highlights

Sarcoidosis is a chronic granulomatous disease that affects many organs, including the lungs
No significant difference was observed in the basal expression levels of PPARγ, ABCA1 or ABCG1 in bronchoalveolar lavage (BAL) cells between C57BL/6J and ABCG1F/F; C57BL/6J mice were used as a wild type control
Consistent with these observations, multiwalled carbon nanotubes (MWCNT) instillation induced fibrosis in myeloidspecific ABCG1 KO mice as indicated by an increase in TGF-β expression by immunofluorescence assay ( Figures 1A,B and S2) as well as trichrome staining of lung tissue (Figure 1C). This increase in TGF-β was observed only in ABCG1 KO mice instilled with MWCNT, suggesting that ABCG1 deficiency enhances the transition from granulomatous inflammation to fibrosis

Summary

Introduction

Sarcoidosis is a chronic granulomatous disease that affects many organs, including the lungs. Previous studies show that both single-walled (SWCNTs) and multi-walled (MWCNT) carbon nanotubes induced pulmonary granulomatous inflammation in animal models. Fujita et al found that SWCNT induces chronic pulmonary granulomatous inflammation [11]; Mercer and colleagues reported that the well-dispersed preparation of SWCNT does not show signs of granuloma formation [12]. Studies from our laboratory demonstrated that oropharyngeal instillation of multiwalled carbon nanotubes (MWCNT) in mice can induce consistent granuloma formation which is stably maintained up to 90 days after instillation and replicates human disease at multiple biological levels with histopathological features and gene expression profiles similar to those observed in sarcoidosis patients [13,14,15]

Methods

Results

Conclusion