title: Probiotics prevent necrotizing enterocolitis by modulating enterocyte genes that regulate innate immune-mediated inflammation.
abstract: Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1β-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.
authors.affiliation: Mucosal Immunology Laboratory, Division of Pediatric Gastroenterology, Massachusetts General Hospital for Children, 114 16th St. (114-3503), Charlestown, MA 02192-4404, USA. kganguli@partners.org
authors.forename: Kriston
authors.initials: K
authors.lastname: Ganguli
authors.forename: Di
authors.initials: D
authors.lastname: Meng
authors.forename: Samuli
authors.initials: S
authors.lastname: Rautava
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
authors.forename: W Allan
authors.initials: WA
authors.lastname: Walker
authors.forename: Nanda
authors.initials: N
authors.lastname: Nanthakumar
mesh_terms: D000818:Animals; D001644:Bifidobacterium / Q000254:growth & development / Q000378:metabolism*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D020345:Enterocolitis, Necrotizing / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D020895:Enterocytes / Q000276:immunology / Q000382:microbiology*; D005786:Gene Expression Regulation; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D018836:Inflammation Mediators / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007421:Intestine, Small / Q000276:immunology / Q000382:microbiology* / Q000637:transplantation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007779:Lactobacillus acidophilus / Q000254:growth & development / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D009924:Organ Culture Techniques; D061251:Primary Cell Culture; D019936:Probiotics; D034622:RNA Interference; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism
chemical_list: C508606:CXCL8 protein, human; D017077:Culture Media, Conditioned; C508600:IL6 protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; D017472:Receptors, Interleukin-1; C546891:SIGIRR protein, human; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C410602:TOLLIP protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4