Immunotoxicity Of Cadimium Telluride Quantum Dots And The Effects On Antigen-presenting Cells

BackgroundQuantum dots(QDs)are nanocrystals with excellent optical properties.The photoluminescence quantum yield of cadmium-containing QDs is 70% higher than that of non-cadmium QDs.Cadmium telluride QDs(CdTe QDs)can emit red fluorescence,which is different from the spontaneous yellow and green fluorescence of tissues,and has a broad prospect in biomedical applications.Although CdTe QDs are becoming increasingly popular in the field of tracer imaging,the safety concerns of their applications are also attracting attention.The immune system is very sensitive to QDs,and understanding QDs’ effects on the immune system is indispensable for QDs to be applied on biomedicine,especially in clinical medicine.The spleen is the main accumulation site of QDs entering the organism and is also an important immune organ.Previous studies have confirmed that very low dose QDs in the spleen could regulate immune response.However,the internal mechanism of this regulatory effect has not been fully elucidated.The immunotoxicity caused by QDs has attracted much attention,especially the effect of QDs on the initiation of immune response and the regulatory effect have not been fully studied.Thus,the underlying molecular mechanism needs to be explored.ObjectivesThis study aims to systematically reveal the immunotoxicity of 3-Mercaptopropionic acid(MPA)-modified CdTe QDs(MPA-CdTe QDs)through model organisms,animals and cell experiments.We want to explore the internal regulatory mechanism,to provide ideas for reducing immunotoxicity for the preparation and modification of MPA-CdTe QDs.We also hope to provide laboratory basis for the clinical application of MPA-CdTe QDs.Contents and methods1.Preparation and characterization of MPA-CdTe QDs.High-quality and stable MPA-modified CdTe QDs were synthesized by electrochemical method,and then characterized by transmission electron microscopy(TEM),UV-visible light spectrophotometry,fluorescence spectrophotometry and Malvern size analyzer.QDs were then characterized by transmission electron microscopy,UV-visible light spectrophotometry,fluorescence spectrophotometry and Malvern size analyzer.2.Investigation the effect of MPA-CdTe QDs on the immune function of the model organism Caenorhabditis elegans(C.elegans).Taking C.elegans as the research object,we investigate the effects of MPA-CdTe QDs on the growth and development and motor behavior of C.elegans,as well as the effects of avoidance behavior and odor aversion learning behavior.We also explored the role of the homologous gene hlh-30(helix-loop-helix transcription)of mammalian transcription factor EB(TFEB)on avoidance behavior.In addition,the damage of MPA-CdTe QDs on four kinds nematode neuron of C.elegans were analyzed.3.To analyze the toxicity of MPA-CdTe QDs on the spleen of mice.The exposure was given by a single injection through tail vein to detect the pathological changes of the spleen at 24 h,7 d and 28 d after injection.The proportion of CD4~+ and CD8~+ T cells in the spleen and peripheral blood,as well as the representative cytokines expressions of Th1,Th2 and Tregs.4.To investigate the effect of MPA-CdTe QDs on antigen presenting cells(APCs).In vitro,THP-1-differentiated macrophages(d THP-1)and human peripheral blood monocyte-derived dendritic cells(MoDCs)were used as research objects to explore the effects of MPA-CdTe QDs-induced TFEB activation on the differentiation and maturation of APCs,and reveal the regulatory pathway of their activation.5.To explore the effect of MPA-CdTe QDs on Th cell activation.MPA-CdTe QDs affected the maturation of MoDCs,and then QDs-exposed DCs were used to mix with T cells in culture,and after in vitro stimulation,the signature molecules of the cells were detected to determine the differentiation of different subsets of Th and the differentiation of Tregs.Results1.Preparation and characterization of MPA-CdTe QDs: The water-soluble MPACdTe QDs used in this study were prepared by mature electrochemical methods.The QDs with an average particle size of 4.7±0.7 nm were obtained by controlling the temperature and duration of the water bath,emitting red fluorescence.TEM results showed that MPA-CdTe QDs were spherical in shape and the particles were evenly dispersed.The results of other optical properties also show that the prepared QDs can meet the experimental requirements of this study.2.The results of C.elegans showed that exposure to 0.01 μmol/L MPA-CdTe QDs enhanced immune genes expression,and 1 μmol/L MPA-CdTe QDs impaired C.elegans’ avoidance-immunity balance while affecting the ability to identify PA14 odor.The molecular mechanisms involved are related to HLH-30 and autophagy activation;the DAF-2/DAF-16 and the MAPK pathways were involved.Moreover,even at the lowest dose of MPA-CdTe QDs exposure caused multiple neuronal damage in C.elegans,this might be another key factor for QDs affecting the immunity.3.The animal studies showed that C57BL/6 mice had no significant tissue damage at different times after single tail vein injection with 0.25 and 2.5 mmol/L MPACdTe QDs,respectively.The results showed that there were no significant differences in CD4~+ and CD8~+ T cells and cytokines expression in various lymphocyte subsets in peripheral blood and spleen.4.MPA-CdTe QDs affect THP-1-derived macrophage differentiation and activation: low concentrations(5 μmol/L)MPA-CdTe QDs reduce the expression of CD11 b,the iconic surface molecule of macrophages,and promote THP-1 cell proliferation.Moreover,the QDs induced macrophage M1 polarization.TFEB activation was also detected.However,the use of TFEB activator(TFEB activator 1)to directly activate TFEB bypassing the mTOR pathway did not promote M1 polarization,which indicating that TFEB activation is a necessary condition for M1 polarization.In addition,we found that inhibiting mTOR using Torin-1 promoted TFEB,but not upregulated CD86.After mTOR activation by MHY1485,QDs-induced TFEB nuclear transfer can be downregulated while CD86 expression can be downregulated,too.By bidirectional regulation of mTOR,we further confirmed that QDs-induced TFEB activation in macrophage polarization participates in the regulation of M1-type differentiation.5.MPA-CdTe QDs affect the differentiation and maturation of MoDCs: low concentrations(5 μmol/L)of MPA-CdTe QDs reduce the expression of CD1 a,a signature molecule on the surface of MoDCs,and hinder the differentiation of monocyte-DCs.Low concentrations of MPA-CdTe QDs reduced the expression of surface co-stimulatory molecules CD86 and HLA-DR of MoDCs,upregulated the expression of tolerant molecules ILT-3 and PDL1,and promoted the differentiation of tolerant DCs(tolDCs).Its intrinsic molecular mechanism is related to TFEB activation,and the mTOR pathway is involved in the regulation of TFEB activation.6.Effects of MPA-CdTe QDs-induced tolDCs on differentiation of CD4+ T cells: Mixed culture of tolDCs and allogeneic T cells promoted Th2 differentiation,inhibited Th1 differentiation.In addition,the results showed the tolDCs promote Tregs differentiation,suggesting that low concentrations(5 μmol/L)MPA-CdTe QDs had immunosuppressive characteristics.ConclusionThis study aims to reveal the influence and regulatory effect of MPA-CdTe QDs on innate immune response.Moreover,it preliminarily explore the effect of MPA-CdTe QDs on Th activation,with a view to speculating about the possible immunomodulatory effects of QDs in clinical applications.Firstly,we found that MPA-CdTe QDs affect C.elegans’ avoidance-immunity homeostasis,and HLH-30 and autophagy activation participate in the regulation.It was found that different concentrations of MPA-CdTe QDs had no significant impairments on the spleen and T cell subsets of C57BL/6 mice at different times by single-time tail vein injection.To explore the mechanisms potentially,we used APCs as the research objects and found that non-lethal doses of MPA-CdTe QDs activate autophagic lysosomes through the mTOR-TFEB pathway,which affecting the development and function of APCs.Then,after mixed lymphatic reaction,the tolDCs induced by MPA-CdTe QDs showed immunosuppressive characteristics.In conclusion,TFEB/HLH-30 activation plays a crucial role in MPACdTe QDs-mediated immunotoxicity,and has the potential to become a research target for immunotoxicity.