Scavenger Receptor Scara3 Of Macrophages Mediates Recognition Of Multi-walled Carbon Nanotubes And Secretion Of Inflammatory Cytokines

Background & Objective Nanomaterials（NM）refer to a general term for ultrafine materials with at least one dimension less than 100 nm in three dimensions.NM has a wide variety of chemical compositions,surface properties,shapes and sizes;according to its composition,it mainly includes metal materials and their oxides,such as nano zinc oxide,titanium dioxide,triiron tetroxide,nano metal tantalum,Nano-alumina,etc.;carbon-based NM comprises fullerene C60,single-walled carbon nanotubes（SWCNT）,multi-walled carbon nanotubes（MWCNT）,carbon black,etc.;there are also some synthetic nanocomposites,for example,polyethylene glycol,polylactic acid,PF14-oligonucleotide nanocomposites,etc.Nanomaterials have excellent performance in many aspects such as small size and unique optical properties,mechanical properties,magnetic properties,diffusion and sintering.It has been widely used in various fields such as electronics,military aerospace,metallurgy,chemical engineering,biomedicine and pharmaceuticals with its unique physical and chemical properties.After entering the ecological environment through atmospheric and biological circulation,NM give rise to some toxic effects to the environment and health.Nanoparticles with extremely small particle size enter the body via the skin,respiratory tract and digestive tract,resulting in direct and /or indirect toxic effects on target tissues and cells.Thus,researches on the toxicity of nanomaterials and mechanisms have attracted more and more attention.The toxicity mechanism of nanomaterials mainly includes the changes of oxidative stress levels,inflammatory reactions and autophagy.Previous animal experiments suggestedthat nano-titanium dioxide promoted tumorigenesis by macrophages-produced CCL3.Also,Silver nanoparticles（AgNP）altered the expression of chemokine 13（CXCL13）and macrophage collagen receptor（MARCO）,which cause damage and neurological disorders of nerve cells.Obviously,recognition and uptake of nanomaterials by macrophages are primarily via their membrane receptors,and are involved in pathogenesis.In the serum,NM easily binds to proteins,forming a so called “protein corona” on the surface.The proteins such as IgG and complements mediate the phagocytosis via FcR and complement receptors on monocytes.However,in the lower respiratory tract and alveoli,where lacks IgG and complements,macrophages are unlikely to mediate their recognition and uptake similar manners.It is possible that macrophages are more likely to recognize NM through membrane-bound pattern recognition receptors（PRRs）.The selective recognition and uptake of PRRs to different nanomaterials have not been elucidated.This current study aimed to explore the role of PRRs of macrophage in mediating recognition of nanomaterials and secretion of inflammatory factors.Methods1.Ultrasonication was used to prepare suspensions of nanomaterials（C60,MWCNT,TiO₂ and SiO₂）.2.To screen and identify PRRs and cytokines with increased expression,the macrophage Raw264.7 cells were stimulated with the four nanomaterials（C60,MWCNT,TiO₂ and SiO₂,the final concentrations were 10 μg/ml）,respectively.After24 hours of stimulation,the total RNA was isolated.Transcriptome high-throughput sequencing was used to and screen PRRs and cytokines stimulated by each kind of NM;qRT-PCR for confirmation of the increased expression of PRRs and cytokines.Then scarvenger receptor Scara3 was obtained.3.To establish Scara3 knockdown（k/d）cell line,Raw264.7 cells were transformedwith the plasmids with shRNA specific for Scara3 by electroporation.Stable k/d cell lines were obtained by G418 selection for 2-3 weeks.k/d efficiency was checked by qPCR and Western-blotting.4.Effects of Scara3 knockdown（1）For analysis effects of Scara3 knockdown on production of inflammatory cytokines,the control cells and Scara3 knockdown cells were treated with MWCNT,and the expression levels of IL-1 β,IL-12 and CXCL9 mRNA were detected by qRT-PCR.The experimental data were analyzed by graph pad1 software t test method.（2）Production of reactive oxygen species in Scara3 knockdown cell line was detected by Flow cytometry and qRT-PCR after stimulation with MWCNT.5.The experimental data were statistically analyzed by t-test using graph pad1 software,and p < 0.05 was statistically significant.Results1.Scara3,IL-1β,IL-12 and CXCL9 were checked as candidate PRRs and cytokines through Transcriptome high-throughput sequencing and screening in Raw264.7 cells treated with four nanomaterials.2.Stable Scara3 knockdown cell lines were successfully established.3.The expression level of IL-1β,IL-12 and CXCL9 mRNA was significantly decreased in Scara3 knockdown cells treated by MWCNT（P<0.05）compared with those of the control cells,while the expression levels of CCL-20,Inhba and Tnfsfl3 b mRNA in Scara3 knockdown cells were not significantly different from those in the control cells（P>0.05）.4.The expression level of reactive oxygen species was significantly decreased in Scara3 knockdown cells treated with MWCNT（P<0.05）compared with that in the control cells.5.The results suggested Scara3 of macrophages was related to the recognition and uptake of MWCNT.ConclusionsThe scarvenger receptor Scara3 was identified to be involved of macrophages uptake and MWCNT recognition by transcriptome high-throughput sequencing and screening,ShRNA knock-down,quantative PCR respectively.Further studies,such as live observation and electronic microscopy of macrophges,are required to confirm the phagocytosis of MWCNT and to investigate its roles in pathogenesis.