Effects Of Trichinella Spiralis Infection On IDO Expression Of Dendritic Cells In Mouse Spleen

Trichinellosis is widespread in the world.Trichinella spiralis(T.spiralis)and its products can affect the immune response of the host,which is conducive to survival in the host.Dendritic cells(DCs)are the most important antigen producing cells(APCs)in the body,and the only APCs that can activate the initial T cells.DCs at different stages can induce different T cells by contact with T cells.Indoleamine 2,3-dioxygenase(IDO)is the only speed limit enzyme outside the liver that catalyzes tryptophan metabolism along the kynurenine pathway,which plays an important role in immune tolerance and is related to immune escape of parasites.The mechanism of IDO on immune escape is not fully unclear in T.spiralis.To dynamically observe the immunomodulatory mechanism of spleen DCs in mice infected with T.spiralis and explore the relationship between IDO mediated immune tolerance and T.spiralis immune escape.The aim is to provide new data for enriching the theory of immune escape and immunosuppression mechanism in T.spiralis.In this study,mice infected with T.spiralis were used as the model,the infection group,PBS control group and 1-Methyl-Tryptophan(1-MT)group were set respectively.Mice were sacrificed at 7 d,15 d,25 d,35d and 50 d after infection.The changes of tryptophan concentration in serum were determined by p-dimethylaminobenzaldehyde colorimetry.The spleen tissues were sliced into paraffin-sectioning,and the pathological changes of spleen were observed by hematoxylin-eosin(HE)staining.Immunohistochemical staining was used to observe the expression of IDO in the spleen.Spleen were taken to prepare single-cell suspension,and the proportion of CD4~+CD25~+cells was detected by flow cytometry(FCM).CD11C~+DCs were sorted by immunomagnetic beads,and the purity of separation was detected by FCM.Total RNA was extracted from the selected cells,q RT-PCR detects the expression levels of different infection time IDO and associated inflammatory factors IL-10,TNF-αand IFN-γmRNA.The expression level of IDO protein in the selected cells was detected by Western blot.The proliferative activity of the sorted cells was detected by Cell Counting Kit-8(CCK-8)assay.The reduction rates of T.spiralis adults and muscle larvae were measured after 1-MT inhibition of IDO.The results of p-dimethylaminobenzaldehyde colorimetry showed that compared with the PBS control group,the tryptophan concentration infected with T.spiralis decreased significantly,and reached the lowest level at the 25th day.Flow cytometry showed a significant increase in the proportion of Tregs in the spleen compared with control mice.HE results showed that the degree of spleen inflammation was the deepest at the 15th day.The boundary between red pulp and white pulp of spleen in PBS control group was clear,the structure of splenic corpuscle and central arteriole was normal,no congestion was observed,and no plasma cell infiltration was observed.After the infection,serious pathological changes occur in the spleen,the degree of inflammation deepens,the inflammation is the deepest in the 15dth degree,as the infection progresses,the spinner’s stimulation to the host decreases,the degree of inflammation gradually decreases.Immunohistochemical results showed that compared with the PBS control group,the expression of IDO in different infection stages of T.spiralis was significantly increased and the expression of IDO was the highest at the 25th day.The q RT-PCR results showed that compared with the PBS control group,the infection group could promote the expression of IDO mRNA in DCs,and the difference was extremely significant.The expression of IDO mRNA reached the highest level at the 25th day,and then began to decrease.Compared with the infected mice,the expression of IDO in 1-MT group was significantly decreased,indicating that 1-MT had a significant inhibitory effect on IDO.Compared with PBS control group,the expression of IL-10 mRNA in spleen DCs in infection group was significantly increased,and the expression of IL-10 mRNA reached the highest level at the15th day.Compared with the infection group,there was no significant change in the expression of IL-10in the 1-MT group,which was not statistically significant.Compared with PBS control group,the expression of IFN-γmRNA in DCs was significantly increased,and the expression of IFN-γmRNA reached the peak at the 7th and 25th day.Compared with the infection group,the 1-MT group was significantly increased on the 15th,25th and 35d,but had no significant change in the other periods.Compared with PBS control group,the expression of TNF-αmRNA in spleen DCs was significantly increased,with statistical significance.The expression of TNF-αmRNA in infected group reached the peak at the 7th and 25th day.Compared with the infection group,the expression of TNF-αin the 1-MT group was significantly different at the 15th day,but had no significant change at other stages.Western blot results showed that the expression of IDO in DCs presented a gradually increasing trend,and reached the peak at the 25th day,and then showed a decreasing trend.The results of CCK-8 method showed that compared with the PBS control group,the OD values of all periods were significantly decreased except for the DCs at the 7th day after infection,indicating that the activity of DCs in the spleen was inhibited at the 7th day after infection.Compared with the infection group,there was no significant change in OD value in the 1-MT group in each period,which was not statistically significant.After 1-MT inhibition of IDO,the number of adult and muscle larvae of T.spiralis in mice decreased significantly,and the reduction rates of adult and muscle larvae were 15.3%and 36.09%respectively.The above results show that T.spiralis infection can induce the high IDO expression of DCs in the host body,and the high expression of IDO will also reduce the concentration of tryptophan,which in turn promotes Tregs proliferation,regulates the host’s immune function,and is conducive to the survival of in T.spiralis the host body.The proliferation activity of DCs in the host body is inhibited during the infection of phithroids,but this inhibition effect is not related to IDO.After T.spiralis infection,the level of inflammatory factor TNF-α,IFN-γand anti-inflammatory factor IL-10 expression increased,however,the expression of IDO can inhibit the expression of inflammatory factors.The initial stage of infection is dominated by Th1 immunity,and the immune response deviates from Th1 to Th2 after day15.At this time the body’s immune response gradually to Th2 type development,which is conducive to the immune escape of the parasite.