Multi-Walled Carbon Nanotubes Induce Arachidonate 5-Lipoxygenase Expression and Enhance the Polarization and Function of M1 Macrophages in vitro
Updated May 22, 2023
NIOSH Dataset RD-1067-2023-0
Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages regulates the production of proinflammatory and pro-resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time-dependent manner. Here we examined the molecular mechanism by which multi-walled CNTs (MWCNTs, Mitsui-7) induce M1 polarization in vitro. Treatment of murine macrophages (J774A.1) with Mitsui-7 MWCNTs increased the expression of Alox5 mRNA and protein in a concentration- and time-dependent manner. The MWCNTs induced the expression of CD68, and that induction persisted for up to 3 days post-exposure. The expression and activity of inducible nitric oxide synthase, an intracellular marker of M1, were increased by MWCNTs. Consistent with M1 polarization, the MWCNTs induced the production and secretion of proinflammatory cytokines tumor necrosis factor-α and interleukin-1β, and proinflammatory LMs leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). The cell-free media from MWCNT-polarized macrophages induced the migration of neutrophilic cells (differentiated from HL-60), which was blocked by Acebilustat, a specific leukotriene A4 hydrolase inhibitor, or LY239111, an LTB4 receptor antagonist, but not NS-398, a cyclooxygenase 2 inhibitor, revealing LTB4 as a major mediator of neutrophil chemotaxis from MWCNT-polarized macrophages. Knockdown of Alox5 using specific small hairpin-RNA suppressed MWCNT-induced M1 polarization, LTB4 secretion, and migration of neutrophils. Taken together, these findings demonstrate the polarization of M1 macrophages by Mitsui-7 MWCNTs in vitro and that induction of Alox5 is an important mechanism by which the MWCNTs promote proinflammatory responses by boosting M1 polarization and production of proinflammatory LMs.
- 1. Quantification of Alox5 mRNA level induced by MWCNTs [XLS – 2 KB]
- 2. Detection of Alox5 protein level induced by MWNTs [XLS – 2 KB]
- 3. Quantification of CD68 and Alox5ap protein expression induced by MWCNTs [XLS – 2 KB]
- 4. Quantification of Nos2 protein expression induced by MWCNTs [XLS – 1 KB]
- 5. Quantitative msmt of proinflammatory cytokines TNF-alpha and IL-1beta.. [XLS – 3 KB]
- 6. Quantitative measurement of proinflammatory LTB4 and PGE2 induced by MWCNTs [XLS – 3 KB]
- 7. Quantification of neutrophil migration induced by MWCNTs [XLS – 2 KB]
- 8. Inhibition of MWCNT-induced proinflammatory LTB4 and PGE2 prodution [XLS – 4 KB]
- 9. Quantification of inhibitors effects on MWCNT-induced neutrophil migration [XLS – 4 KB]
- 10. Quantification of Alox5 and CD68 protein in Alox5 knockdown [XLS – 3 KB]
- 11. Quantitative measurement of proinflammatory LTB4 in Alox5 knockdown [XLS – 1 KB]
- 12. Quantification of neutrophil migration in Alox5 knockdown [XLS – 2 KB]
- Data Dictionary [PDF – 139 KB]
- Detailed Methods [PDF – 157 KB]
Data Collection Methods
- Characterization of nanoparticles
- MWCNTs (Mitsui-7) were prepared in a control medium (CM) [Dulbecco’s modified eagle’s medium (DMEM) with 1% fetal bovine serum (FBS)] at a concentration of 2 mg/ml through vortex and by sonication.
- Carbon black (CB, Printex 90) was prepared in a control medium (CM) like MWCNTs and used as carbon-based particle control.
- Stock solutions of MWCNTs or CB were further diluted with the culture media for MWCNTs at 2.5 or 10 µg/ml or for CB at 2.5, 10, or 30 µg/ml, sonicated immediately before use, and were treated for indicated time.
- Cell culture, polarization, and treatment
- J774A.1 murine monocyte/macrophage cells were grown in DMEM with 10% fetal bovine serum.
- J774A.1 cells (5 x 105 cells/ml) were seeded in DMEM with 3% FBS for 1 day and then treated with other reagents for 1 day or 3 days.
- M1 polarization was induced with interferon γ (20 ng/ml) plus lipopolysaccharides (100 ng/ml) and M2 polarization was induced with interleukin 4 (20 ng/ml) for indicated time (typically three days).
- Control media (media with 1% FBS) were prepared and treated to establish a negative control response.
- HL-60, a promyelocytic cell line, was grown in the RPMI1640 medium with 10% FBS.
- Differentiated HL-60 cells (dHL-60) were prepared with use of 2 µM all-trans retinoic acid (ATRA) treatment for 3 days.
- Quantitative real-time PCR (RT-qPCR)
- Detection and quantification of Alox5 or β-actin at mRNA level.
- Real-time qPCR was performed, and the relative expression change values were calculated as 2−ΔΔCt and expressed as fold change in comparison with untreated control.
- Detection and quantification of Alox5, CD68, Alox5ap, or β-actin.
- Images were scanned using HP scanjet and were used to quantify each band with ImageJ software.
- Detection of nitric oxide synthase 2 (Nos2, mouse iNOS) expression
- Detection and quantification of Nos2 expression using an intracellular Nos2 detection assay kit.
- The fluorescence signal was measured using a fluorescence plate reader and the relative fluorescence unit (RFU) was expressed.
- Enzyme-linked immunosorbent assay (ELISA)
- Production of proinflammatory cytokines (TNF-α, IL-1β) and lipid mediators (LTB4, PGE2) in cell-free culture supernatant were measured using ELISA kits with a microplate reader equipped with SOFTmax PRO 4.0.
- Chemotaxis assay
- Transwell cell migration assay was performed using a cell migration assay kit to determine the effect of MWCNTs on neutrophilic cell migration in vitro.
- ATRA-induced differentiated HL-60 (dHL-60) cells were collected and suspended in RPMI 1640 medium without FBS (2.5×105 cell/well/100 µl) and plated on each upper chamber.
- The lower chambers were filled with cell-free culture supernatants, indicated amount of MWCNTs in the basal RPMI 1640 medium containing 1% FBS, or RPMI 1640 medium containing 10% FBS.
- For inhibition assay, a cell-free culture media from cells treated with a specific cyclooxygenase 2 inhibitor, NS-398 (at 2 or 10 µM), a leukotriene A4 hydrolase inhibitor, Acebilustat (at 1 or 5 µM), or dimethyl sulfoxide (DMSO) as a vehicle 6 hr prior to MWCNTs or IFN-γ+LPS exposure added at each lower chamber. A specific LTB4 receptor inhibitor LY293111 (5 or 25 nM) or DMSO as a vehicle was added directly into the dHL-60 cell suspension.
- The fluorescence signal was measured using a fluorescence plate reader and the relative fluorescence unit was expressed to show the migrated cells.
- Alox5 gene silencing
- To knockdown Alox5 gene expression in macrophages, mouse Alox5-specific short hairpin (shRNA) as RNA interference was introduced into macrophages and incubated for 2 days and then cells were used for RT-qPCR and immunoblotting analysis.
- Cells were treated with MWCNTs or M1 inducer and subsequently used for RT-qPCR and immunoblotting analysis, and the cell-free culture media was collected and used for ELISA assay.
Lim CS, Veltri B, Kashon M, Porter, DW, and Ma Q (2023) Multi-Walled Carbon Nanotubes Induce Arachidonate 5-Lipoxygenase Expression and Enhance the Polarization and Function of M1 Macrophages in vitro. Nanotoxicology 17(3): 249-269. doi.org/10.1080/17435390.2023.2204161
This work was funded by the NORA Program under Grant 9390BMX (Q.M.) and the NTRC program under Grant 9390HTN (Q.M.), at the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, USA.
Chol Seung Lim1 (email@example.com)
Brandon Veltri2 (firstname.lastname@example.org)
Michael Kashon3 (email@example.com)
Dale W. Porter4 (firstname.lastname@example.org)
Qiang Ma1,5 (email@example.com)
1 Receptor Biology Laboratory, Toxicology and Molecular Biology Branch, HELD, NIOSH, Morgantown, WV 26505, USA
2 Department of Microbiology, Immunology, and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26505, USA
3 Bioanalytics Branch, HELD, NIOSH, Morgantown, WV 26505, USA
4 Pathology and Physiology Research Branch, HELD, NIOSH, Morgantown, WV 26505, USA
5 Corresponding author