Multi-walled carbon nanotubes induce arachidonate 5-lipoxygenase expression and enhance the polarization and function of M1 macrophages in vitro

Abstract

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.

Keywords:

• Pulmonary inflammation
• MWCNT
• macrophage polarization
• Alox5
• LTB4

Acknowledgements

The authors thank S. Friend for assistance in TEM imaging of MWCNTs and carbon black.

Author contributions

Q.M., D.W.P., and C.S.L. conceived the study; Q.M., D.W.P., and C.S.L. designed the study; C.S.L. and B.V. performed experiments; C.S.L. and M.K. performed statistical data analyses. C.S.L. provided a draft manuscript. Q.M. finalized the manuscript. All authors contributed to manuscript preparation.

Disclosure statement

No potential conflict of interest was reported by the author(s). The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention.

Additional information

Funding

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.