| Trichinellosis is a common zoonotic parasitic disease that is caused by the ingestion of raw or poorly cooked meats containing the infective larvae of Trichinella spiralis(T.spiralis).T.spiralis exerts an immunomodulatory effect on the host immune response through its excretory secretion(ES)product.T.spiralis and its products can inhibit the host immune response,which is conducive to the survival of the parasite in the host.Dendritic cells(DCs)are classified as professional antigen-presenting cells(APC)with high effectiveness that perform multiple functions in the immune system,such as the secretion of cytokines and other immune function molecules.Indoleamine 2,3 dioxygenase(IDO)is a potent immunoenzyme present in DCs,interactsing with certain exogenous mediators(antigens,etc.)to participate in immune evasion.However,the immune escape mechanisms in which immune tolerance of DCs induced by ES antigen of T.spiralis and the role of IDO in immune escape of T.spiralis remain unclear.To explore the mechanism in IDO induced by T.spiralis ES antigens stimulating DCs,we set up the model of bone marrow-derived DCs in mice.DCs were exposed to ES antigen,IFN-γ,1640 medium and the blank control.After treated with various stimulants,the expressions of IDO protein on the surface of DCs were detected by immunofluorescence technique.The expressions of cell surface markers such as CD40,MHC-Ⅱ,CD80 and CD86 on DCs were detected by flow cytometry.The m RNA expression levels of IDO and cytokines such as IL-10,TNF-α and IFN-γ were determined by real-time quantitative PCR(q RT-PCR)at different time points.The protein expression level of IDO was detected by Western blot.Transferred with s i RNA620,the m RNA expression levels of IDO and its relative downstream cytokines in DCs were detected by q RT-PCR.Meanwhile,the protein expression level of IDO was detected by Western blot.According to IDO,IFN-γ,TNF-α,IL-10 and β-actin sequence,real-time PCR primers were designed and analysed to establish a fluorescence real-time PCR detection method.The results of cell proliferation activity(CCK-8)showed that there was no effect on the activity of DCs in the concentration of ES antigen at 0~25 μg/m L.In the results of q RT-PCR,the m RNA expression of IDO in DCs was different in various ES antigen concentration and stimulant time,with a dose and time-dependent effect in partly period of time.The expression of IDO m RNA was increased within 0~24 h.The m RNA expression level of IDO was significantly highest at 24 h compared to 0 h.Therefore,the T.spiralis ES antigen could promote the maximum m RNA expression of IDO on 15 μg/m L concentration at 24 h.The results of IDO protein level detection were consistent with the q RT-PCR,and we chose a 15 μg/m L T.spiralis ES antigen concentration as the working concentration.The results showed ES antigen can efficiently promote the expression of IDO in DCs cytoplasm in cellular immunofluorescence experiments.The surface molecules CD40,MHC-Ⅱ,CD80 and CD86 of DCs were increased at different extents stimulated by ES an tigen and lipopolysaccharide(LPS)by flow cytometry.LPS could significantly promote the high expression of surface molecules in DCs,with significantly different from the ES antigen group(P<0.001).It was proved that LPS could promote the maturation and enhance the immune activity of DCs.After the action of ES antigen,the expression of surface molecules of DCs is not significant.The maturation of DCs was inhibited,leaving it in a semi-mature state,which restrained the antigen presentation ability of DCs and promoted immune escape of the worms.The results of q RT-PCR showed that there was a linear relationship between the m RNA expression of IDO and the time of DCs stimulated by 15 μg/m L of ES antigen in the 0~24 h period.The m RNA expression of IDO increased continuously with time.The expression of IDO m RNA gradually decreased and reached to equilibrium finally during the 24~72 h period.The ES antigen significantly promoted the m RNA expression of the proinflammatory cytokines TNF-α,IFN-γ and anti-inflammatory cytokine IL-10 downstream of IDO in DCs.The results of Western blot showed that the expression of IDO protein in DCs increased continuously at 0~36 h after ES antigen treatment while gradually decreased after 36 h,and reached to equilibrium finally.Transfected with si RNA620 and treated with 15 μg/m L of ES antigen for 36 h in DCs,the m RNA level of IDO and downstream cytokines were detected by q RT-PCR again.The results showed that si RNA620 can effectively inhibit the level of target genes of IDO,with significant difference compared to the single ES antigen group(P<0.001).Due to si RNA620 interference,the m RNA levels of the downstream pro-inflammatory cytokines TNF-α,IFN-γ and anti-inflammatory cytokine IL-10 were significantly increased,which was opposite to the undisturbed group.Western blot analysis showed almost no expression of IDO protein in si RNA620 group,which was significantly different from that of single ES antigen group(P<0.001).From all results above,it confirmed that the expression of the co-stimulatory molecules MHC-Ⅱ,CD80,CD40 and CD86 were upregulation after DCs stimulated by T.spiralis ES antigen.Moreover,the m RNA and protein level of IDO were increased significantly.The expression of pro-inflammatory factors TNF-α,IFN-γ and anti-inflammatory cytokine IL-10 were upregulated.Inflammatory response were activated and produced immune tolerance of DCs,which in turn,the T.spiralis was allowed to escape from the body and survived in the host,resulting in a persistent chronic infection of the host.It is suggested that sustained stimulation of DCs by ES antigens may induce DCs remaining immune tolerant through activating IDO-mediated inflammation relative factors.This research not only establishes a model for studyin g the immunoregulatory mechanism of nematodes in vitro,but also provides new evidence and ideas for the clinical treatment of trichinosis and the development of effective vaccines. |
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