Studes On The Immunomodulatory Effects Of Trichinella Spiralises Es Antigen And Hsp70on Dcs

Trichinellosis is a common important foodborne zoonotic disease. Humans and other mammals become infected mainly due to ingestion of raw or undercooked meat contained Trichinella spiralis larvae cyst. In the whole life cycle, the excretory-secretory antigen(ES) of Trichinella spiralis plays a key role in the invasion and persistent infection to the host. HSP70is one of the most highly conserved proteins among all living creatures, which can participate in evading host immune function by activating macrophages and dendritic cells. But the mechanism of ES antigen and HSP70activate or inhibit the body’s immune is unclear.With the model of DC2.4in mice, this study observed the activated ability of ES antigen and HSP70on DC2.4. Then ES antigen stimulated repeatedly and LPS/ES antigen s stimulation after the ES antigen’s stimulation firstly, and HSP70stimulated repeatedly and Pam3csk4/HSP70’s stimulation after the HSP70’s stimulation firstly, respectively. The expression levels of TLR4/TLR2, transcription factor NF-κB and AP-1were detected by real-time PCR, and also related proteins and cytokines level were determined by Westen blot and ELISA, respectively. Surface molecules CD80/CD86were also observed by flow cytometry.According to TLR2, TLR4, NF-κB, AP-1and GAPDH sequence, real-time PCR primers were designed and analysised to establish a fluorescence real-time PCR detection method. Cell Counting Kit-8assay results showed that concentration of ES antigen and HSP70within25μg/ml will not affected the vitality of DCs. As the different ES antigen concentration and action time, the TLR4mRNA expression was different with certain limits on dose and action time, while with the increase of the concentration and time, the amount of TLR4mRNA expression has a tendency to reduce. We chose15μg/ml and12h as the optimum concentration and time, respectively. As to HSP70, the TLR2mRNA expression took on a similar way, so we chose5μg/ml and12h.Immune tolerance study of DCs showed that ES antigen and LPS (TLR4agonist) activated by raising obviously the level of surface molecules CD80/CD86, increasing dramatically the mRNA expression TLR4, NF-κB and AP-1also the protein expression of MyD88, NF-κB and c-jun and p38, promoting significantly the inflammatory cytokines IL-6, IL-12, NO and TNF-a level also anti-inflammatory cytokines IL-10. After activation, as the DCs were stimulated by LPS/ES antigen again, compared with DCs’s activation, we come to the conclusion that the drop levels of surface molecules CD80/CD86, the lessened mRNA expression TLR4, NF-κB and AP-1, the lower protein expression MyD88, NF-κB, c-jun and p38, the reduced levels of inflammatory cytokines IL-6, IL-12, NO and TNF-a and anti-inflammatory cytokines IL-10.HSP70and Pam3csk4(TLR2agonist) activated by raising obviously the level of surface molecules CD80/CD86, increasing dramatically the mRNA expression TLR2, NF-κB and AP-1also the protein expression of MyD88, NF-κB, c-jun and p38, promoting significantly the inflammatory cytokines IL-6, IL-12, NO and TNF-a level also anti-inflammatory cytokines IL-10. After activation, as the DCs were stimulated by LPS/ES antigen again, compared with DCs’s activation, we come to the conclusion that the drop levels of surface molecules CD80/CD86, the lessened mRNA expression TLR4, NF-κB and AP-1, the lower protein expression MyD88, NF-κB, c-jun and p38, the reduced levels of inflammatory cytokines IL-6, IL-12, NO and TNF-a and anti-inflammatory cytokines IL-10.Above results confirmed that under a certain dose and time, the ES antigen and HSP70can raise surface molecules CD80/CD86, induce the expression of TLR2and TLR4, activate downstream NF-κB and MAPK signal pathway, induce the secretion of inflammatory cytokines, The DCs activated by ES antigen or HSP70after activation, with the results of CD80/CD86expression downgrading, inhibition of the expression of TLR2and TLR4and downstream signal pathway, all of these resulting in immune tolerance on DCs. It was showed that the constant stimulation of ES antigen and HSP70or the PPRs cross stimulus were much more likely induced a similar immune tolerance throμgh TLR2/TLR4mediating the NF-κB and MAPK pathway of DCs which was induced by endotoxin previously. All of these not only provided successfully the the immunomodulatory mechanism of research about worm in vitro model, but also a new basis and train of thought for clinical treatment and effective vaccine about trichinosis.