Investigation Of The Ability Of 4,4’-Diaponeurosporene Producing Bacillus Subtilis In Enhancing Piglet Gut Mucosal Immune Function

Piglet diarrhea caused by various pathogenic microorganism is one of the most severe infection diseases among pig farms.In recent years,the outbreak of piglet diarrhea has resulted in heavy economic losses in China.The mucosal immune system aplasia of piglet is one of the main reasons of diarrhea.Therefore,promoting mucosal immune system development will be benefit for preventing diarrhea.Recently,more and more studies show that carotenoids have the ability to promote immune organs development and enhance immunity.Besides,Bacillus subtilis(B.subtilis)have a well-established safety record and have been explored as a feed additive.Combining the beneficial effects of carotenoids and B.subtilis offered us a new approach to enhance mucosal immunity.So,firstly,we constructed a series of plasmids containing different carotenoids synthases or dehydrogenases genes,and transformed into B.subtilis.The carotenoid in transformants was extracted and identified by HPLC;Secondly,we compared the influences of B.subtilis produced carotenoid(Dia)with β-carotene on the progress of dendritic cell(DC)maturation,antigen uptake,and migration.We also investigated the role of signal molecules and CD36 in Dia induced DC maturation;Thirdly,we tested the influence of B.s-Dia(B.subtilis that could produce Dia)on mouse mucosal immunity though experimental Salmonella typhimurium infection,and investigated the cell and molecular mechanisms underlied this progress.Finally,we detected the effect of B.s-Dia on mucosal immune system of piglets.Our study laid the foundation of the application of B.s-Dia as a mucosal immune enhancer.Our research is divided into the following four parts in detail:1 Construction of carotenoid producing B.subtilisWe constructed a series of plasmids containing different carotenoids synthases or dehydrogenases genes,such as crtY,crtl,crt B,crtE,crtM,and crtN,and transformed into B.subtilis.The transformant harboring pMK3-Pcrtm-crtMN(B.s-Dia)produced carotenoid.After extracted by acetone-hexane,the carotenoid was identified by HPLC.The result showed that the carotenoid produced by B.subtilis was C30 carotenoid,4,4’-diaponeurosporene(Dia).After serial passage,B.s-Dia still had the ability to produce Dia.The obtaining of highly purified Dia laid the foundation for further studying the biological function of Dia.In addition,genetically stable B.s-Dia provided basis for fermentation production of Dia.2 The effects of Dia on DCThe carotenoids,a subfamily of the isoprenoids,are among the most widespreadnatural substances,and play important biological roles as accessory light-harvesting components of photosynthetic systems,photoprotecting antioxidants,and regulators of membrane fluidity.Acoording to the molecular structure,carotenoids can be divided into two categories,C30 carotenoids and C40 carotenoids.The immune functions of many C40 carotenoids have been extensively researched.While as another kind of important carotenoids,C30 carotenoids were seemingly ignored from the beginning.In this study,the functions of Dia,a C30 carotenoid,on DCs maturation were identified for the first time.The results indicated that Dia was a potent inducer of DCs maturation via CD36 and NF-κB signals.Dia-treated DCs had a stronger ability to stimulate Thl polarization.However,β-carotene had opposite effects on DCs phenotypic maturation and reduced IL-12p70 production.These findings provided new insight into the different immune functions of carotenoids.Moreover,these findings shed light on a previously unappreciated function of carotenoid,and laid a foundation for further application.3 The effects of Dia on mouse mucosal immunityIn our previous study,we found Dia is a potent inducer of DC maturation in vitro,and could stimulate Thl polarization.However,whether B.s-Dia could improve mucosal immune was unclear.So,we investigated the influence of B.s-Dia on mouse mucosal immune.We found that oral administration of B.s-Dia improved mice mucosal immunity as showed by an increased resistance against S.typhimurium infection.Further study showed that B.s-Dia administration increased CCL20 secretion by intestinal epithelial cells,recruiting more dendritic cells into lamina propria.Moreover,the number of Th17 and Treg was also increased.CD36 was necessary to induce CCL20 secretion and blocking its signaling with specific antagonist,Sulfo-N-succinimidyl Oleate(SSO)compromised the immunoenhancement ability of B.s-Dia.In addition,supplementation of B.s-Dia reduced the number of CD8αα~+ intraepithelial lymphocytes(IELs),probably resulted from the decrease of IECs derived TGFβ.These results preliminarily indicated the beneficial outcome of B.s-Dia on mucosal immunity.4 The effects of Dia on piglet mucosal immune systemThis study investigated the influences of B.s-Dia on piglet mucosal immune system.Oral administration of B.s-Dia remarkably improved the development of Peyer’s patch(PP)and crypt depth,and the number of goblet cell was also increased.Moreover,B.s-Dia also increased the number of IEL,while B.s had no significant influence on it.In addition,B.s-Dia also increased the number of IgA~+ cells,CD4~+ and CD8~+ cells in ileum lamina propria.These results indicated that B.s-Dia possessed a better property to promote porcine mucosal immune system development,and might serve as an immunopotentiator candidate.