The Endotoxin Tolerance Immune Regulatory Effects Of Paternal Dietaryastragalus Polysacchirides On Broiler Chickens

Breeding and intensive production can enhance the production performance of broiler chickens,but it is harmful to their immunity,and could lead to high-frequency infection and environment stress.Especially during the first half of development(from day 1 to day 21),broiler chicken is more likely to have infectious disease,and injured more than adul t chicken.Immune system of chicken has distinct developmental and immunity characteristic at different developmental stages.And so,the genes transcriptome,as well as promotor methylation of core regul ator in thymus were analyzed to learn the thymus immunity at different developmental stages.This experimental work will play vital role in guiding the immune modul ation of chicken.Astragalus membranaceus(APS),is a kind of immune-modul ator extracted from dry root of radix astragali,which can be used as an immune-modul ator to modul ate the immunity of animals.APS coul d moderately activate the immune system and promote the immunity and growth performance of animals.To learn the transgenerational regul atory effects of APS,we carried out a survey to study the effect of paternal dietary APS supplementation on gene transcription of themselves and broiler chickens,also to explore how APS affect the immunity of their off-springs.Experiment 1 Age-related changes on immune gene expression and immunology function of chicken thymus120 one-day-old breeder cocks,included 15 cages of 8 birds each,were fed to learn the aging's effect on chicken's thymus immunity.At 2(2-W)and 40(40-W)weeks of age,one chicken each cage was randomly chosen and slaughtered to get the thymus sample.The resul ts showed that thymus weight and morphology of 40-W group were far different from that of2-W group,and exhibited a property of degeneration.Considering this phenotype variances,we analyzed the thymus'transcriptome to investigate the molecul ar mechanism that had been implicated in this phenotype diversity with age.Pearson correlation coefficients and principal component analysis indicated that two major popul ations corresponding to 40-W and 2-W group were identified,and 1949 differentially expressed genes(DEGs,1822up-regul ated and 127 down-regul ated)were obtained.Resul ts of GO and KEGG pathway enrichment found that 4 significantly enriched KEGG pathways(Cytokine-cytokine receptor interaction,Intestinal immune network for IgA production,Toll-like receptor(TLR)signaling pathway,AGE-RAGE signaling pathway in diabetic complications)related to immunoregul ation were screened between 40-W and 2-W group.These resul ts confirmed that thymus immunity of chickens had strong age-related correlation.DEGs related to these4 enriched KEGG pathways were suppressed in thymus of 2-W group,this indicated that thymus immunity of 2-weeks-age chick was suppressed.CD40 is involved in 3 of the 4significantly enriched pathways,and it is critical for thymus immune-regul ation.CD40promotor methylation level of 2-W group was higher than that of 40-W group,it is consistent with the transcriptional differences of the gene.Our study concluded that thymus immunity of chicken was vary with age.Compared to the 40-W group,thymus immunity of2-W group was suppressed on the whole,and these effects might be related to CD40suppression induced by promotor hyper-methylation of the gene.Experiment 2 Effects of dietary astragalus polysaccharides on jejunal mucosal immunity of breeder cocksCompared to grown chicken,TLR signaling pathway was suppressed in thymus of chicken at young age,which will further affect the immunity of broiler chicken.APS,as a highly branched heteroglycan,or acid hydrolyzed into oligosaccharides,neither can be absorbed through intestinal mucosa into the body.To learn the nutri-epigenetics role of Astragalus polysaccharides(APS),we designed and studied the transgenerational effect of paternal dietary APS supplementation in chickens.160 Avein breeder cocks were randomly allocated into 5 groups and fed with 0,0.01,0.1,1,and 10.00 g/kg APS supplement,respectively.At 44 weeks of age,one fertile breeder cock each cage were choosen and slaughtered to get tssue samples,including serum,intestinal mucosa,and spleen sample.It showed that the paternal dietary 10.00 g/kg APS coul d transgenerational promote growth performance and jejunal tissue morphology of chickens.As to phenotypes,10.00 g/kg APS treatment was chosen for molecul ar assays.According to the resul ts of in vitro antibacterial test,both bacterial inhibition ring test and agar plate diffusion method showed that APS did not have any direct antibacterial effect.It meant that the effect of APS was probably not due to bacterial inhibition,but rather interacted directly with intestinal immune system to modul ate the bird immunity.The resul ts of in vivo test showed that,in jejunal mucosa,10 g/kg dietary APS didn't have any systematic effect on gene transcription of breeder cocks.As well,the APS had no effects on cytokine levels in serum of breeder cocks.In spleen of breeder cocks,the dietary APS didn't have any systematic effect on TLR4 signaling pathway regul ators,cytokines,and endotoxin tolerance related genes transcription.Our data revealed that the dietary APS supplementation coul d significantly promote the intestinal tissue morphology of breeder cocks.However,on account of long-term adaption,the APS had no effect on immunity of breeder cocks.Experiment 3 Transgenerational effects of paternal dietary astragalus polysaccharides on immunity of broiler chickensAs a resul t of long-term adaptation,the dietary APS additive has no significant effect on intestinal mucosal immunity or spleen immunity.In this test,we analyzed the effects of paternal dietary APS on growth performance and immunity of broiler chickens.160one-day-old Avein breeder cocks were used in a single-factor design with 0,0.01,0.1,1,and10.00 g/kg APS,respectively,4 replicated cages each group,and 8 birds each cage.When the breeder cocks at 40-week-age,sperm of breeder cocks was collected and used for hatching experiment to get broiler chickens when the cocks at 40-weeks-old.Sperm of the fertile cock was collected,and used to fertilize the corresponding 12 hens.After 3 times insemination in five days,20 fertilized eggs(weight between 65 and 70 g)each replicate were collected and used for hatching experiment to get offspring chickens.About 16offspring chickens each replicate were got,the offspring chickens were grouped according to their fathers'diet,fed a commercial pelleted diet without APS supplementation,and had fresh water at libitum.The chickens were weighed daily to portray the growth curve from 1to 14 days of age.Then,in each group,the four fertile cocks(one breeder cock each cage)used for mating and 12 randomly selected offspring chickens(14 days old,3 chickens each cage)were slaughtered by cervical dislocation to get and analyze the intestinal tissue morphology.We also collected serum,intestinal mucosa,and spleen samples.We found that paternal dietary APS coul d transgenerational promote the growth performance and jejunal tissue morphology of broiler chickens.And the paternal dietary APS coul d induce transgenerational ET-like effect through activating the IFN?-SOCS1 pathway in broiler chickens.During this process,the two down-stream pathways employed by TLR4 showed binary opposition property.TRIF pathway was activated,whereas MyD88 pathway was not affected.The cytokines,type I interferons lie downstream of TRIF were up-regul ated.And further more,type I interferons woul d trigger IFN?-SOCS1 pathway and induce SOCS1-dependent ET-like immune responses.And the intestinal mucosal immune system coul d regul ate the systemic immunity through the circul atory system.The paternal dietary APS coul d transgenerational up-regul ated the serum type-I-interferon level of offspring chickens,significantly.Just like intestinal immune system,the paternal dietary APS supplementation coul d also induce endotoxin tolerance-like immune response(TLR4pathway)in spleen of broiler chickens.In this process,the APS activated the TRIF pathway,but suppressed the MyD88 pathway.Moreover,the paternal dietary 10.00 g/kg APS coul d significantly increase the transcription level of TNF-?,IL-6,and IL-4(as well as GATA3).But the APS had no significant effect on transcription of pro-inflammatory cytokines and ET related regul ators.This means that the paternal dietary APS can transgenerational induce endotoxin tolerance-like immune response in spleen,and the fundamental cause of this response might lies on its effect on intestinal mucosal immunity.Through its immune regul atory effects,the paternal dietary APS coul d transgenerational improve the growth performance and jejunal tissue morphology of broiler chickens.Experiment 4 Effects of astragalus polysaccharides on TLR4 signaling pathway in Caco2 cellTLR signaling pathway is the most important immunological recognition related pathway.According to the resul ts of experiment 1,we found that,in comparison to grown chicken,TLR pathway in thymus of 2-week-old chick was in a status of hypo-activition.17core regul ators(10 DEGs and 7 other regul ators)of TLR signaling pathway were all significantly down-regul ated in thymus of chick.As an important part of TLR signaling pathway,TLR4 pathway was also suppressed.And we can improve the immunity of2-week-old chick by moderately activating the immune system.As an efficient immunoregul ator,APS coul d moderately activate TLR4 signaling pathway of intestinal mucosal immune system,and further optimize the animals'immunity.In this experiment,we analyzed the immunology effects of APS on Caco2 cell.The Caco2 cell was used in a single-factor design with APS or LPS treatment,including the control group,APS(APS,1 mg/mL),LPS group(LPS,1 ug/mL),APS and LPS group(AL),and LPS induced endotoxin tolerance group(ET),and each group had 3 replications.We analyzed the transcription and translation of TLR4 signaling pathway regul ators,cytokines,and endotoxin tolerance related regul ators by using qRT-PCR and flow cytometry,respectively.We also tested the synthesis and secretion of proinflammatory cytokines(IL-1,IL-8,and TNF-?).The resul ts showed that LPS coul d activate inflammatory response of TLR4-MyD88 signaling pathway,and significantly(P?0.05)increase the expression of proinflammatory cytokines(IL-1,IL-8,and TNF-?),which woul further significantly(P?0.05)promote the synthesis and secretion of IL-8.While,APS coul d inhibit the inflammatory response induced by LPS,and induce an endotoxin tolerance like immune response.TLR4-TRIF siganaling pathway played a vital role in this process.And then,we learned the immunology effects of APS and LPS on Caco2 cell in the context of TRIF inhibited by hs-siRNA.We found that,when the TRIF was significantly(P?0.05,about 70%knockdown)suppressed,APS coul d not successful ly inhibit the inflammatory response induced by LPS.The expression pattern of TLR4 signaling pathway and related cytokines inTRIF~-and AL group was very similar to the LPS treatment group.Above all,we coul d conclude that APS coul d effectively inhibit the LPS-induced inflammatory response by activating TRIF related endotoxin tolerance response,and significantly decrease the expression level of proinflammatory cytokines.The resul ts of this experiment further confirmed that the transgenerational immune regul atory effects of APS were derived from its affection on intestinal mucosal immune system.Experiment 5 Epigenetic modifications are essential for the paternal immunomodul atory effects of APSTo learn the nutri-epigenetics mechanism of paternal dietary APS,we collected the jejunal mucosa,spleen,and sperm,and analyzed the promoter methylation and histone modification of MyD88,TRIF,and SOCS1.The experimental resul ts showed that the paternal dietary APS coul d significantly(P?0.05)reduced the promoter methylation level of SOCS1 in jejunal mucosa of breeder cocks and sperm.The SOCS1 promoter methylation in jejunal mucosa of broiler chicken,as well,was also lower(but not significantly)in the APS treatment group.The transcription factors with high-affinity binding activity in SOCS1gene promoter region were all transcription activators.So,the APS induced hypo-methylation coul d promote the transcription of SOCS1.And hypo-methylation in promoter region of SOCS1 had a certain correlation with the transgenerational effects of paternal dietary APS.However,the paternal dietary APS had no significantly effect on the promoter methylation of MyD88 and TRIF,nerther on the promoter methylation of SOCS1in spleen.The methylation level of MyD88,TRIF,and SOCS1 in spleen exhibited the property of tissue-specific hypo-methylation.The paternal dietary APS coul d also significantly(P?0.05)affect the histone methylation of MyD88 and TRIF in jejunal mucosa and spleen of breeder cocks and broiler chicken.In addition,the paternal dietary APS coul d significantly(P?0.05)increase the Dimethyl-H3K9 level of MyD88,and decrease(P?0.05)the Dimethyl-H3K9 level of TRIF in sperm.In brief,DNA methylation(SOCS1)and histone modification(MyD88 and TRIF)are critical for the paternal immune-regul atory effects of APS.Conclusion:In summary,our study concluded that thymus immunity of chicken was vary with age,and thymus immunity of 2-W group was suppressed on the whole.And,in Caco2 cell,APS coul d induce TLR4-TRIF pathway related endotoxin tolerance response.By this way,the APS coul d also targeted improve the immunity of broiler chicken.While,through transgenerational nutri-epigenetic regul atory mechanism,the paternal dietary APS supplementation coul d induce transgenerational endotoxin tolerance-like effect in jejunum mucosa of broiler chickens,and indirectly induce endotoxin tolerance-like immune response in spleen.In this way,the paternal dietary APS supplementation coul d transgenerational promote the growth performance and intestinal morphology of broiler chickens at young age.In a word,the APS additive to breeder cocks coul d improve the immunity and growth performance of broiler chickens through nutri-epigenetics mechanism.And in this way,APS can deal with the immune-suppression problem of young-age chicken.