Screening Of Bifidobacterium Bifidum With The Ability To Induce IL-10 In Vitro And Its Immunomodulatory Effects In Vivo

Interleukin-10(IL-10)is an important inflammatory and immunosuppressive cytokine.In recent years,some specific strains or mixtures of intestinal microorganisms,including Lactobacillus,Bifidobacterium and Bacteroides,have been reported to be able to up-regulate the level of IL-10 in both in-vivo and in-vitro models,and significantly alleviate the inflammation associated diseases.Typical models used to evaluate IL-10 inducing abilities of probiotics include human peripheral blood mononuclear cell(PBMC)model,macrophage model and so on.But a low efficacy and a relative weak mimic for intestinal immune context hinders their effectiveness in strain screening.Im et al.reported a co-culture model of mesenteric lymph node(MLN)and screened out the anti-inflammatory Bifidobacterium bifidum(B.bifidum)PRI1 in 2008.Based on their reports,this study employed their in-vitro co-culture model,and carried out a high-throughput IL-10 inducing probiotic screening of lactic acid bacteria.Both bioactive components and underlying mechanisms of typical high inducing strain were further explored.The in-vivo inducing abilities in both germ-free and SPF mice as well as its roles in TNBS-induced colitis were also evaluated to provide theoretical basis for its further application.In the present study,probiotics with IL-10 inducing abilities were screened by in vitro model,and its effective components and underlying mechanisms were studied.Firstly,the co-culture in-vitro model of MLN was established in our laboratory according to the literature reports.The level of IL-10 in the supernatant after co-culture of bacteria and MLN cells was determined and the effects of anti-CD3 and anti-CD28 on the production of IL-10 were compared.The results showed that both Lactobacillus and Bifidobacterium could not induce the production of IL-10 without any stimulants or only anti-CD3.After stimulated with anti-CD3 and anti-CD28,Lactobacillus could not induce production of IL-10 while specific Bifidobacterium could induce IL-10 production with the highest level of IL-10 more than400pg/m L.This study shows that neither Bifidobacterium nor Lactobacillus can directly induce the production of IL-10 in the in-vitro model.In the state of immune activation,specific B.bifidum induce the secretion of IL-10.Based on this,an in-vitro MLN evaluation model for IL-10 production induced by Lactic acid bacteria was established.Thereafter,210 strains of Bifidobacterium were screened.The results showed that the induction ability of B.bifidum could be divided into three levels: high,medium and low(or without dectable IL-10).Due to the significance of B.bifidum in anti-inflammation function and probiotic industry,87 strains of B.bifidum isolated and preserved in our laboratory were further evaluated,and B.bifidum B96 was screened as one of the most promising strains.Further studies showed that inducing ability of B96 was dose-dependent and thermosensitive.After a protease treatment,the ultrasonic supernatant of bacterial suspension could no longer induce IL-10 production,and the extraction of bacterial outer membrane protein and total protein could not induce a considerable amount of IL-10.It is speculated that the surface lipoprotein or glycoprotein of B.bifidum B96 may be involved in the induction of IL-10 production by MLN.The results of RNA-sequencing(RNA-seq)showed that B.bifidum B96 enhanced the adpative immune response induced by anti-CD3 and anti-CD28 and activated the expression of antigen presentation and costimulatory molecules,inducing the balalce of Th1 and Th2 to a incline of Th2 and the balalce of Th17/Tregs to a incline of Tregs.Further analysis showed that inducing abilities of B.bifidum B96 is dependent on Toll-like receptor(TLR)2 rather than TLR4.After intragastric administration of B.bifidum B96 in germ-free,SPF and TNBS-induced colitis mice,the levels of IL-10 in the colon tissue,T regulatory cells(Tregs),survival rate and inflammatory associated index in mice were measured.The results showed that B.bifidum B96 could up-regulate the level of Tregs in the lamina propria of colon of GF mice,but had no effect on the level of Tregs in MLN and spleen,and there was no significant difference in the level of IL-10 in colon.In homeostasis,B.bifidum B96 had no effect on the number of Tregs in MLN and spleen as well as the level of IL-10 in colon in SPF mice.However,intragastric administration of B.bifidum B96 could significantly alleviate colitis induced by TNBS.When detected after 5 days of modeling,the survival rate of B96 group was 100%,there was no obvious colonic damage,and the level of colon cytokines was matched to the control group.This anti-inflamamtory effect of B96 was not due to the modulation role of B96 in gut microbiota composition,even though gavage of B.bifidum B96 significantly increased the abundance of Bifidobacterium.