| Avian coccidiosis is a protozoal disease caused by intracellular protoz oan Eimeria influncing the development of the poultry industry seriously.E.tenella is the most pathogenic among several Eimeria species,which can cause intestinal lesions and hinder the chickengrowth and development.Exploring and understanding the pathogenesis of chicken coccidia,and clarifying the innate immune response mechanism of anti-coccidial infection is meaningful for the prevention and treatment of the disease.Chicken Toll-like receptor 15(Ch TLR15)is a kind of avian-specific pattern recognition receptor located on the cell surface.At present,its ligand and activation mechanism are still unclear.Ch TLR15 plays an important role in chicken intestinal pathogen infection,but the mechanism is not clear.The role of Ch TLR15 in intestinal damage caused by chicken coccidia infection has not been reported.The NOD(Nucleotide-binding oligomerization domain)-like receptor 3(NLRP3)inflammasome plays a vital role in mediating the inflammatory response is assembled by sensor protein(NLRP3),adaptor protein(ASC)and Caspase.It was thought that NLRP3 activation is closely related to pathogenic bacteria,but recent reports have suggested that NLRP3 also plays an important role during infection of intracellular parasites.Up to now,the activation mechan ism of NLRP3 in the process of coccidiosis infection was not reported.Considering NLRP3 is an endogenous molecule,pathogens cannot directly activate it.Whether or not inflammatory responses and injuries caused by coccidiosis infection are relate to NLRP3,and whether avian-specific Ch TLR15 is involved in coccidiosis infection has not been reported till now.In this study,both E tenella infection experiments in vivo and in vitro were conducted to explore the dynamic changes of related molecules in Ch TLR15 and NLRP3 signal pathway after E.tenella infection.In addition,we also elucidated the specific activation of Ch TLR15 by E.tenella sporozoites and the correlation between Ch TLR15 and NLRP3 signal pathway.(1)The gene fragment of Ch NLRP3-600 including 600 base pairs were ligated into the prokaryotic expression vector p ET-30a(+)and construct the recombinant plasmid p ET30(a)-Ch NLRP3-600.The objective plasmid was transferred into E.coli BL21 and the expression of the protein was detected by SDS-PAGE after the positive bacteria were induced by IPTG.New Zealand white rabbits were immunized with the expressed protein to prepare polyclonal antibodies against Ch NLRP3-600 protein.The antibody titer was determined by indirect ELISA and the reactivity of the polyclonal antibody was detected by Western blot.The results showed that Ch NLRP3 wasexpressed as insoluble inclusion bodies in E.coli,and the molecular weight was about 27 k Da.The titer of the polyclonal antibody detection was 217.Western blot results showed that the prepared rabbit anti-Ch NLRP3 polyclonal antibody could react well with chicken cecum tissues protein.In addition,New Zealand white rabbits were immunized with the Ch TLR15 polypeptide fragment to prepare a Ch TLR15 polyclonal antibody,which showed a titer of 217 and a good specific reaction with chicken cecum tissues protein.Specific antibodies to rabbit-derived Ch NLRP3 and Ch TLR15 provided the necessary materials for subsequent detection.(2)To clearly identify the expression levels of each molecule contained in the pathway Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Ch Caspase-1/Ch IL-1?/Ch IL-18 in chicken cecal tissues,E tenella infection experiments in vivo was carried on..SPF chickens were orally infected by low dosage(3×104)or high dosage(5×103)of E.tenella sporulated oocysts.Cecal tissues of challenged chickens were sampled at 4,12,24,72 h post infection.Quantitative real-time PCR(q RT-PCR)was used to detect the transcription levels of each molecule in pathway Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Ch Caspase-1/Ch IL-1?/Ch IL-18,and Western Blot and indirect ELISA were used to detectthe protein expression levels of Ch TLR15,Ch NLRP3 and Ch IL-1? in cecum tissues.The results displayed that the expression of each of the above molecules in the cecal tissue from infectedchickens was significantly up-regulated within 72 hours post E.tenella infection(p<0.001),and the dynamic changes were generally consistent.These results indicated that Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Ch Caspase-1/Ch IL-1?/Ch IL-18 signaling pathway plays an role in coccidiosis infection.(3)To further determine the roles and relationships of the above molecules in the innate immune response against chicken coccidiosis infection,the transcriptional level of each molecule in Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Caspase-1/Ch IL-1?/Ch IL-18 pathway and the protein expression of Ch TLR15,Ch NLRP3 and Ch IL-1? in DF-1 cells at 0,2,4,6 and 8 h post E.tenella sporozoites stimulation was detected in vitro.It was found that E.tenella sporozoites stimulated DF-1 cells in vitro significantly up-regulateed the expression levels of each molecule(p<0.001),and the dynamic changes of each molecule are consistent with time,and reach the peak at 8 h post sporozoite stimulation.The results showed that the E.tenella sporozoite can activate the signaling pathway of Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Caspase-1/Ch IL-1?/Ch IL-18.(4)The RNA intereference(RNAi)technique was used to interfere expression of Ch TLR15 in DF-1 cells.The results of optimizing the transfection con ditions of Ch TLR15 si RNA showed that the final concentration of 50 n M of Ch TLR15 si RNA effectively interfered the expression of Ch TLR15.The transcriptional levels of each molecule in Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Caspase-1/Ch IL-1?/Ch IL-18 pathway and the protein expression of Ch TLR15,Ch NLRP3 and Ch IL-1? in Ch TLR15-low-expressed DF-1 cells at 0,2,4,6 and 8 h post E.tenella sporozoites stimulation were detected.The results showed that the expression of related molecules contained in the above pathway was all significantly up-regulated compared to the negative si RNA transfected cells but nott sporozoites-stimulated group(p<0.001),and the expression levels were significantly lower than that of negative si RNA transfected cells with sporozoite-stimulated group(p<0.001),and the dynamic tendency of each molecule was essentially consistent.In addition,the Ch TLR15 gene fragment was cloned into the eukaryotic overexpression vector p CMV,and the positive recombinant plasmid p CMV-Ch TLR15 was constructed.The target positive plasmid was transfected into DF-1 cells.Western Blot detection found that the expression of Ch TLR15 protein was significantly upregulated at 36 h posttransfection with p CMV-Ch TLR15(p < 0.001).The transcriptional levels of each molecule contained in Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Caspase-1/Ch IL-1?/Ch IL-18 pathway and the protein expression of Ch TLR15,Ch NLRP3 and Ch IL-1? in Ch TLR15 overexpressed-DF-1 cells at 0,2,4,6 and 8 h post E.tenella sporozoites stimulation was detected.The results showed that the expression of each molecule was significantly higher than that of p CMV transfected with no sporozoite-stimulated group and p CMV transfected and with sporozoite-stimulated group(p<0.001),and the dynamic tendency of each molecule was basically consistent.The results clearly indicated that the E.tenella sporozoite can specifically activate the Ch TLR15/Ch My D88/Ch NF-?B signaling pathway,then further activated the Ch NLRP3/Ch Caspase-1/Ch IL-1?/Ch IL-18 pathway.The Ch TLR15/Ch My D88/Ch NF-?B/Ch NLRP3/Ch Caspase-1/Ch IL-1?/Ch IL-18 pathway plays a vital role in the early intestinal inflammatory response and injury caused by coccidiosis infection.In conclusion,E.tenella can specifically activated Ch TLR15 and then activated the key related molecules Ch My D88 and Ch NF-?B.The activated Ch TLR15 further activated Ch NLRP3 and the pathway-related molecules Ch Caspase-1 to participate in the body's innate immune response against chicken coccidia infection,and induces the large production of the factors Ch IL-1? and Ch IL-18 to mediate the early inflammatory.A cross-talk existed between Ch TLR15 and the Ch NLRP3 signaling pathway.This study provides a theoretical basis and reference for the development of a new generation of anticoccidial preparations based on signaling pathway inhibitors. |
PDF Full Text Request