| Objective:Arsenic(As)is a naturally occurring element that can be found in various sources and can enter the human body through multiple routes.Exposure to arsenic is a major public health concern as it can adversely affect the immune system and has the potential to cause cancer in different tissues of the body.Additionally,inorganic arsenic can impair the immune defense function of the body and lead to various chronic health issues.Despite these known effects,the precise molecular mechanisms underlying arsenicinduced immune system damage are not yet fully understood.Further,there is a dearth of in-depth research investigating how inorganic arsenic heightens susceptibility to infectious diseases and triggers abnormal immune responses.It is therefore imperative to conduct extensive fundamental research to elucidate these issues.Dendritic cells(DCs)are crucial in the immune system as they act as specialized antigen-presenting cells(APCs),playing a vital role in pathogen surveillance,autoimmune tolerance,transplant rejection/survival,and tumor immunity for both the innate and adaptive immune response.However,research has shown that inorganic arsenic has detrimental effects on DCs.Arsenic exposure can disrupt the expression of molecules on the membrane surface of DCs and impair their cytokine secretion.Furthermore,it may weaken the ability of DCs to track,phagocytose,degrade,and transport pathogens to T cells,resulting in immunosuppressive effects.The effects of arsenic exposure on DC migration and cross-antigen presentation have not been extensively studied.Recent advances in autophagy research have uncovered its involvement in various immune response processes,including maturation,migration,cytokine secretion,antigen presentation,and T cell activation in DCs.As a result,autophagy is closely linked to innate and adaptive immune responses in DCs,in both physiological and pathological contexts.Our previous studies have demonstrated that exposure to inorganic arsenic affects the production of autophagic vesicles in bone marrow-derived dendritic cells(BMDCs).Therefore,exploring the impact of inorganic arsenic on autophagy and its potential role in suppressing immune function in DCs is crucial.Autophagy is a physiological process that primarily relies on lysosomal degradation.Lysosomes play a vital role in various physiological activities and cellular homeostasis in the human body,as the final degradation organelle in the complete autophagic flux process,as well as a central organelle.It is important to investigate the possible effects of inorganic arsenic exposure on autophagic flux and lysosomal function in DCs,as well as the role of the autophagy-lysosome system in the inorganic arsenic suppression of immune function in DCs.Transcription factor EB(TFEB)and transcription factor E3(TFE3)are members of the MiT/TFE family of proteins,which have gained cosiderable attention in recent years as crucial transcription factors regulating the autophagy-lysosome pathway.In particular,TFEB plays a major role in lysosomal biogenesis and autophagy regulation,making it a central regulator of the autophagic process.However,it is unclear whether the lysosomal system can impact lysosomal and autophagic dysfunction in inorganic arsenic-exposed BMDCs.Given the intimate relationship between the autophagy-lysosome system and innate immunity,immune tolerance,and immunogenicity of DCs,it is worth investigating whether TFEB can modulate phagocytosis,degradation,and antigen presentation to T cells in arsenic-exposed BMDCs.Further investigation is needed to determine whether TFEB can mitigate the effects of immune damage such as phagocytosis,degradation,and antigen presentation to T cells in arsenic-exposed BMDCs.TFE3,like TFEB,is a member of the MiT/TFE family and shares similar tissue expression patterns and biological functions.Recent research has highlighted its potential role as an important regulator of autophagy and lysosomal homeostasis in immune system activation,allergic disease control,and expression of critical metabolic regulators.It remains unclear whether TFE3 can also modulate the autophagy-lysosome system and immune function similarly to TFEB.Therefore,further investigation is necessary to elucidate the potential regulatory functions of TFE3 in these biological processes.In summary,this study investigated the impact of inorganic arsenic exposure on autophagic vesicle formation and autophagic flux in BMDCs through in vitro extraction and cultivation of primary BMDCs.The study also investigated key factors that may affect autophagic flux,such as lysosome-related functions.Subsequently,the study utilized knockdown and overexpression gene editing techniques to establish TFEB/TFE3 knockdown and overexpression BMDC cell models to evaluate the potential impact of these key transcription factors of the autophagy-lysosome pathway on lysosomal number,lysosomal-associated proteins,lysosomal acidic environment,LMP,and autophagy-related proteins in arsenic-exposed BMDCs.Finally,the study explored the potential effects of TFEB/TFE3 knockdown and overexpression on phagocytosis,the expression of membrane surface molecules and receptors(including antigen-presenting molecules,co-stimulatory molecules,adhesion molecules,and chemokine receptors),cytokine secretion,migration capacity,and MHCI-like cross-antigen-presenting function in arsenic-exposed BMDCs.Methods:1.To investigate how inorganic arsenic exposure affects autophagic flux and lysosomal function in dendritic cells,we isolated and cultured primary BMDCs.These cells were then divided into three groups:control group(Con),arsenic group(1 μM AsⅢtreatment for 6,12,and 24 h),and LPS-treated arsenic-stained group.To ensure the purity of the primary BMDCs,we tested them by flow cytometry.We extracted total and lysosomal proteins from the primary BMDCs and used Western blot to detect the expression levels of various proteins,including LC3II/LC3-I,p62,Atg5,Atg12-Atg5,Atg16L1,LAMP1,LAMP2,CTSD,CTSL,CTSB,CTSS,and AEP.Additionally,we used RT-PCR to detect the mRNA expression of key proton pump-related components of lysosomal acid regulation.To investigate the fusion of autophagosomes with lysosomes,the fluorescence intensity of Gal3 and LAMP1,and the distribution of CTSB,we used immunofluorescence.We also used Lyso-Tracker live cell staining to detect the number of lysosomes,Lyso-Sensor live cell staining to detect the lysosomal pH value,and AO staining to detect lysosomal membrane permeability.2.To explore the role of TFEB/TFE3 in regulating lysosomal function and autophagic flux in dendritic cells under inorganic arsenic exposure,we employed CRISPR/Cas9 knockdown technology to construct a TFEB/TFE3 knockdown plasmid(TFEB/TFE3 KD)and a negative control plasmid(NC,TFEB/TFE3 scrambled).We also utilized exogenous gene overexpression technology to construct a TFEB/TFE3 overexpression plasmid(TFEB/TFE3 OE)and a negative control plasmid(NC,TFEB/TFE3 scrambled).To establish TFEB/TFE3 knockdown and overexpression BMDCs cell models,electrotransfection was performed.Western blot analysis was used to measure the expression levels of TFEB,TFE3,LAMP1,LAMP2,CTSD,CTSL,CTSB,CTSS,LC3II/LC3-I,p62,and Atg5.Furthermore,RT-PCR was also performed to measure the mRNA expression of Tfeb,Tfe3,and lysosomal acidic regulatory key proton pump-related components.Immunofluorescence was performed to detect Gal3 and LAMP1 fluorescence intensity,while Lyso-Tracker live cell staining was used to assess the number of lysosomes.In addition,Lyso-Sensor live cell staining was used to determine the pH of lysosomes.3.Investigation of the regulatory role of TFEB/TFE3 in the suppression of dendritic cell immune function by inorganic arsenic exposure.The phagocytic ability of BMDCs was observed using fluorescence microscopy with FITC-OVA and fluorescent latex beads.The expression of antigen-presenting molecules in BMDCs was determined by flow cytometry.Expression of co-stimulatory molecules,adhesion molecules,chemokine receptors and cytokines in BMDCs was analysed by RT-PCR,while cytokine secretion was measured by ELISA.Migration ability was assessed by Transwell,cross-antigen presentation by ELISA and β-galactosidase.Results:1.Effects of inorganic arsenic exposure on autophagy flux and lysosome of primary BMDCs.1.1 Inorganic arsenic blocks autophagy flux of primary cultured BMDCs.Inorganic arsenic exposure can induce the expression of autophagosome marker protein LC3-II/LC3-I and the upstream key autophagy-related gene Atg5 in BMDCs,suggesting that arsenic exposure may induce the activation of autophagy-related genes and increase the production of autophagosomes.In addition,arsenic exposure increased the expression of autophagy substrate protein p62,suggesting that the degradation of autophagosomes in BMDCs may be inhibited after arsenic exposure,and inorganic arsenic may inhibit the process of autophagy flux in primary BMDCs.The results of the study using BMDCs treated with rapamycin and CQ indicate that the increase in LC3-II/LC3-I caused by arsenic exposure is not only due to autophagy activation but also to insufficient LC3-II/LC3-I degradation.Both arsenic exposure and 3MA intervention significantly raised the levels of p62,suggesting that arsenic may lead to p62 accumulation,which is a substrate of autophagy.Inorganic arsenic exposure not only triggers the production of primary BMDCs autophagosomes,but it also disrupts the autophagy process,leading to an accumulation of autophagy substrate proteins due to the inadequate degradation of autophagosomes.1.2.Effects of inorganic arsenic exposure on fusion of autophagosome and lysosome in primary cultured BMDCs.After arsenic exposure,there was no observed blockage of fusion between autophagosomes and lysosomes in BMDCs.1.3.Effects of Inorganic arsenic exposure on lysosome and its function in Primary cultured BMDCs.As the exposure time to arsenic increased,the fluorescence intensity of the LysoTrackerRed labeled lysosomes in primary BMDCs continued to decrease.This suggests that arsenic exposure may decrease the number of acidic lysosomes present.Additionally,the expression of lysosomal membrane proteins and tissue proteolytic enzymes such as LAMP1,LAMP2,CTSD,CTSL,CTSB,CTSS,and AEP were significantly induced by arsenic exposure.A study showed that prolonged exposure to arsenic resulted in a decrease in the green fluorescence intensity of Lyso-SensorGreen,a lysosomal pH probe.In addition,RT-PCR results showed that the expression of Atp6v0c,Atp6v0e and Atp6v1a was reduced by arsenic exposure.These results suggest that the acidic environment of primary BMDCs lysosomes can be damaged by arsenic exposure.After conducting AO staining experiments,it was discovered that exposure to arsenic may lead to an increase in lysosomal membrane permeabilization(LMP)in BMDCs.The results also revealed that as the duration of arsenic exposure increased,there was a gradual rise in the fluorescence of Gal3 in primary BMDCs,and the number of Gal3 fluorescence sites overlapping with LAMP1 also increased.These results strongly suggest that exposure to inorganic arsenic elevates the lysosomal LMP of primary BMDCs.After exposure to arsenic,Western blot analysis revealed a significant decrease in the expression of CTSD and CTSB proteins in lysosomes.In addition,the co-localization of CTSB and LAMP1 was significantly reduced.These results suggest that arsenic exposure may alter the distribution of cathepsin in lysosomes of BMDCs and lead to cathepsin leakage in lysosomes of BMDCs.2.The possible role of TFEB/TFE3 in the effect of inorganic arsenic exposure on the function of BMDCs lysosome and autophagy flux.2.1.Effect of TFEB/TFE3 on the expression of TFEB and TFE3 in BMDCs exposed to arsenic.TFEB/TFE3 knockout and overexpression models were constructed in primary BMDCs.After treatment with 1 μM AsⅢ,it was found that the corresponding TFEB and TFE3 protein levels in TFEB/TFE3 KD+As treatment group were significantly lower than those in NC+As group,while the corresponding TFEB and TFE3 protein levels in TFEB/TFE3 OE+As group were significantly higher than those in NC+As group.Primary BMDCs were used to establish knockout and overexpression models of TFEB/TFE3.It was observed that the levels of TFEB and TFE3 proteins were significantly reduced in the TFEB/TFE3 knockout group treated with As.Conversely,the TFEB/TFE3 overexpression group treated with As showed significantly increased levels of TFEB and TFE3 proteins.2.2.Effect of TFEB/TFE3 on lysosome and its function of BMDCs exposed to arsenic.To begin with,in the TFEB/TFE3 knockout model,the Lyso-Tracker fluorescence intensity was notably lower in both the TFEB/TFE3 KD and TFEB/TFE3 KD+As treatment groups compared to the NC group and NC+As group.Upon overexpression of TFEB/TFE3,the decrease in Lyso-Tracker fluorescence intensity induced by arsenic exposure was observed to be restored.Intervention with TFEB/TFE3 on lysosomal related proteins in BMDCs exposed to arsenic showed that the protein levels of LAMP1,LAMP2,cathepsin CTSD,CTSL,CTSB and CTSS in TFEB/TFE3 knockout group and TFEB/TFE3 KD+As group were significantly lower than those in NC group and NC+As group.After overexpression of TFEB/TFE3,it was found that the protein levels of LAMP2,CATD,CTSL,CTSB and CTSS in BMDCs before and after arsenic exposure were significantly increased.The fluorescence intensity of Lyso-Sensor in the TFEB/TFE3 KD+As group showed a significant decrease compared to the NC+As group.Overexpression of TFEB/TFE3 after arsenic exposure demonstrated a significant increase in the fluorescence intensity of LysoSensor.These results suggest that TFEB/TFE3 knockout may exacerbate the detrimental effects of arsenic exposure on the lysosomal acidic environment,whereas overexpression of TFEB/TFE3 may alleviate these effects.After knockout of TFEB/TFE3 in BMDCs,the lysosomal LMP was exposed to inorganic arsenic,resulting in increased fluorescence intensity of Gal3 in both the TFEB/TFE3 KD+As group compared to the NC+As group.Conversely,overexpression of TFEB/TFE3 resulted in a significant decrease in the fluorescence intensity of Gal3 in the TFEB/TFE3 OE+As treatment group compared to the NC+As group.These results suggest that TFEB/TFE3 knockout may exacerbate lysosomal LMP following arsenic exposure,whereas TFEB/TFE3 overexpression may alleviate this increase.2.3.Effect of TFEB/TFE3 on autophagy of BMDCs exposed to arsenic.In the TFEB/TFE3 knockout model,the specific knockout sequence of TFEB significantly reduced the protein expression of LC3-Ⅱ/LC3-Ⅰ,p62 and Atg5 after arsenic exposure compared to the NC+As group.Conversely,when TFE3 was downregulated by its specific knockout sequence,the expression levels of LC3-Ⅱ/LC3-Ⅰ and p62 proteins were further increased in the TFE3 knockout group compared to the NC+As group.Overexpression of TFEB/TFE3 significantly increased the protein level of the autophagy marker protein LC3-Ⅱ/LC3-Ⅰ and decreased the protein level of the autophagy substrate p62 compared to the NC group.In the TFEB/TFE3 OE+As group,the protein level of LC3-Ⅱ/LC3-Ⅰ was significantly increased,while the protein level of the autophagy substrate p62 was significantly decreased compared to the NC+As group.These results suggest that overexpression of TFEB/TFE3 can alleviate the blockage of autophagic flux in BMDCs induced by arsenic exposure.3.Possible regulatory role of TFEB/TFE3 in the inhibition of immune function of primary BMDCs by arsenic exposure.3.1.Effect of TFEB/TFE3 on phagocytosis of BMDCs exposed to arsenic.After exposure to arsenic,it was discovered that the fluorescence intensity of soluble FITC-OVA in primary BMDCs decreased significantly,as well as its phagocytosis of latex beads.This suggests that arsenic exposure can reduce the phagocytosis of primary BMDCs.Furthermore,in comparison to the NC group,the TFEB/TFE3 knockout group showed a significant reduction in fluorescence intensity of FITC-OVA and phagocytosis of latex beads.On the other hand,overexpression of TFEB/TFE3 led to a significant increase in phagocytosis of latex beads.3.2.Effect of TFEB/TFE3 on BMDCs antigen presentation molecules exposed to arsenic.In BMDCs induced by LPS,compared with NC group and NC+As group,TFEB/TFE3 knockout decreased the expression of MHC I and MHC II in primary BMDCs before and after arsenic exposure,and increased the expression of MHC Ⅰ and MHC Ⅱ in TFEB/TFE3 OE+As group compared with NC+As group.3.3.Effects of TFEB/TFE3 on costimulatory molecules,adhesion molecules,chemokine receptors and cytokines of BMDCs exposed to arsenic.In BMDCs induced by LPS,the mRNA expressions of Cd70,Cd80,Cd83,Icam1,Ccr7,Tnf-α,Il-6 and Il-12b in TFEB/TFE3 knockout group were significantly lower than those in NC group.Compared with NC+As group,the mRNA expression of Cd80,Cd83,Icam1,Ccr7,Tnf-α,Il-1β,Il-6 and Il-12b decreased significantly in TFEB/TFE3 KD+As group.When TFEB/TFE3 was overexpressed,the mRNA expression levels of Cd40,Cd80，Cd83,Cd86,Icam1,Ccr7,Tnf-α,Il-1β,Il-6 and Il-23a in TFEB/TFE3 overexpression group were significantly higher than those in NC group.Compared with NC+As group,the mRNA expression of Cd86,Icam1,Ccr7,Tnf-α,Il-1β,Il-23α and Il-6 increased significantly in TFEB/TFE3 OE+As group.3.4.Effect of TFEB/TFE3 on the migration ability of BMDCs exposed to arsenic.With or without LPS-induced differentiation,arsenic exposure significantly decreased the migration ability of BMDCs.After LPS induced differentiation of BMDCs,compared with NC group and NC+As group,TFEB/TFE3 knockout decreased the migration ability of BMDCs before and after arsenic exposure.After overexpression of TFEB/TFE3,the migration ability of primary BMDCs in TFEB/TFE3 OE+As treatment group was significantly higher than that in NC+As group.3.5.Effect of TFEB/TFE3 on cytokine secretion of BMDCs exposed to arsenic.In the primary BMDCs induced by LPS,the content of TNF-α,IL-1β,IL-6 and IL-10 in the culture supernatant of TFEB knockout group was significantly lower than that of NC group,while the content of IL-1β in the culture supernatant of TFEB overexpression group was significantly increased.Compared with NC+As group,the content of IL-1β in the culture supernatant of TFEB KD+As group decreased significantly,while the content of TNF-α and IL-1β in cell culture supernatant increased significantly in TFEB OE+As group.Compared with NC group,the content of TNF-α and IL-1β in cell culture supernatant of TFE3 knockout group decreased significantly,while the content of IL-6 and IL-10 in cell culture supernatant of TFE3 overexpression group increased significantly.Compared with NC+As group,the content of IL-1β and IL-10 in the culture supernatant of TFE3 KD+As group decreased significantly,while the content of TNF-α and IL-6 in cell culture supernatant increased significantly in TFE3 OE+As group.3.6.Effect of TFEB/TFE3 on cross-antigen presentation function of BMDCs exposed to arsenic.The secretion of cytokine IL-2 in the supernatant of one-way mixed culture decreased significantly after exposure to inorganic arsenic,and the activity of β-galactosidase in hybridoma cells of B3Z CD8+T cells was significantly decreased after exposure to arsenic.The results showed that arsenic exposure could decrease the cross antigen presentation function of primary BMDCs.Compared with NC group,both TFEB/TFE3 knockout and overexpression could significantly reduce the activity of β-galactosidase in B3Z CD8+T hybridoma cells.However,knockout or overexpression of TFEB/TFE3 had no significant effect on βgalactosidase activity among all groups of BMDCs exposed to arsenic.Conclusion:Arsenic exposure has been shown to impair autophagic flux in dendritic cells(DCs)by impairing lysosomal function,a critical component of this process.As a primary regulatory transcription factor of the autophagy-lysosome pathway,knockdown of TFEB/TFE3 exacerbates the impairment of lysosomal function and autophagic flux in DCs exposed to arsenic.Conversely,TFEB/TFE3 overexpression can alleviate arsenic exposure-induced lysosomal impairment,autophagic flux blockade and immune function in DCs.This suggests that inorganic arsenic inhibits lysosomal function in DCs,which in turn inhibits autophagic flux.However,TFEB/TFE3 promotes lysosomal biogenesis and increases the expression of autophagy-related genes in DCs,thereby activating the autophagy-lysosome system and providing protection against inorganic arsenic inhibition of cellular immune function in DCs. |
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