Effects Of Four Inhibitory Molecules From Eimeria Maxima On IL-12 Secretion Of Chicken Macrophages And Dendritic Cells

Coccidiosis is a parasitic protozoosis caused by single or mutiple species of Eimeria.Eimeria maxima(E.maxima),one of the most prevalent species,results in great impacts on the growth and feed utilization of chickens,causing great economic losses to the poultry industry worldwide.After invadeing the gut of chicken,chicken coccidia stimulates a series of immune reactions including humoral immunity and cellular immunity.Cellular immunity plays a key role in the immune response against coccidia.In the early stage of chicken coccidiosis infection,IL-12 participates in the initiation of cellular immunity,the key immune response against chicken coccidiosis.IL-12 can promote the production of IFN-γ secreted by NK cells and T cells,and then stimulate the differentiation of Th1 cells,which play an important role in the anti-coccidiosis of chicken.The inhibition of Th1 cell differentiation is conducive to the survival of protozoa in the host.It is an important means for intracellular protozoa to escape from the host’s immune clearance.Innate immunity is the first defense line against pathogen invasion.Once pathogen associated molecular patterns(PAMPs)are recognized by pattern recognition receptors(PRRS),the innate immune response is activated.PRRS,especially Toll like receptors(TLRs),are mainly expressed by dendritic cells and macrophages,and play an important role in initiating immune response against pathogens.In addition,it has been reported that many protozoa can interfere with IL-12 secretion by regulating key molecules in cytokine related signaling pathways(such as NF-κ B,ERK1/2,p38,JNK,STAT3).In our previous study,we had screened four inhibitory molecules from E.maxima c DNA library,namely Em SAG,Em HPSP-1,Em HPSP-2 and Em HPSP-3,which inhibited the expression of Th1 cell specific transcription factor T-bet and IFN-γ,IL-2 cytokines,indicating that they may play a role in the immune escape of coccidia.However,what is the mechanism of these inhibitory molecules in innate immunity?What signaling pathways are involved? Can Th1 cell differentiation be inhibited by regulating innate immune molecules? None of these questions is clear.Therefore,in order to clarify these problems,this paper made the following research.In order to detect the change of chicken Toll like receptors(ch TLRs)transcription level induced by four E.maxima inhibitory molecules.Chicken macrophages and chicken bone marrow-derived dendritic cells(ch BMDCs)were treated with above inhibitory molecules(Em SAG,Em HPSP-1,Em HPSP-2 and Em HPSP-3)respectively.The m RNA levels of ch TLR-1,ch TLR-2,ch TLR-3,ch TLR-4,ch TLR-5,ch TLR-7,ch TLR-15 and ch TLR-21 were detected by quantitative real-time PCR(q RT-PCR).The results showed that the four inhibitory molecules significantly inhibited the m RNA levels of ch TLR-2,ch TLR-4 and ch TLR-21.Subsequently,in order to detect the effect of four E.maxima inhibitory molecules on cytokines secretion in chicken macrophages and ch BMDCs.Chicken macrophages and ch BMDCs were treated with four inhibitory molecules.Transcription levels of IL-12,TNF-α,IL-10 and TGF-β were detected by q RT-PCR.The expression of IL-12 was determined by ELISA(enzyme-linked immunosorbant assay).The results showed that the four inhibitory molecules significantly down-regulated the transcription and expression level of IL-12 in chicken macrophages and ch BMDCs.Furtherly,in order to determine the related signaling pathways of IL-12 secretion in chicken macrophages.q RT-PCR and western blot were used to detect the changes of key molecules involved in the signal pathways of IL-12 secretion(NF-κ B,ERK1/2,p38,JNK,STAT3).Finally,RNAi was used to verify the immune function of the key molecules in the signaling pathway.The results revealed that the expression of ERK1/2phosphorylated protein was significantly up-regulated by the four inhibitory molecules.Knockdown of ERK1/2 gene significantly reduced the inhibitory effect of four E.maxima inhibitory molecules on IL-12.These results indicate that the four inhibitory molecules can inhibit the secretion of IL-12 by up-regulating the expression of ERK1/2phosphorylated protein,which is the key molecule in ERK-MAPK pathway.This study explored the effect of four E.maxima inhibitory molecules on ch TLRs of chicken macrophages and ch BMDCs.Subsequently,the effects of four inhibitory molecules on IL-12 secretion and related signaling pathways of chicken macrophages were observed as well.The results showed that the four E.maxima inhibitory molecules(Em SAG,Em HPSP-1,Em HPSP-2 and Em HPSP-3)inhibited the m RNA transcription levels of ch TLR-2,ch TLR-4 and ch TLR-21,and significantly inhibited the expression of IL-12.The inhibition of IL-12 expression was achieved by up-regulating the phosphorylation of ERK1/2.It is of great significance to clarify the mechanism of E.maxima inhibitory molecules on chicken macrophages and ch BMDCs,and it is of great reference value for eluciding the immune escape and innate immune mechanism of coccidiosis,and also provides theoretical basis for the prevention of coccidiosis.