title: Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG.
abstract: BACKGROUND
  Probiotic bacteria are increasingly used as immunomodulatory agents. Yet detailed molecular knowledge on the immunomodulatory molecules of these bacteria is lagging behind. Lipoteichoic acid (LTA) is considered a major microbe-associated molecular pattern (MAMP) of Gram-positive bacteria. However, many details and quantitative data on its immune signalling capacity are still unknown, especially in beneficial bacteria. Recently, we have demonstrated that a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having modified LTA molecules, has an enhanced probiotic efficacy in a DSS-induced colitis model as compared to wild-type.
  
  
  RESULTS
  In this study, the importance of D-alanylated and acylated LTA for the pro-inflammatory activity of LGG was studied in vitro. Purified native LTA of LGG wild-type exhibited a concentration-dependent activation of NF-κB signalling in HEK293T cells after interaction with TLR2/6, but not with TLR2 alone. Chemical deacylation of LTA interfered with the TLR2/6 interaction, while a moderate effect was observed with chemical dealanylation. Similarly, the dltD mutant of LGG exhibited a significantly reduced capacity to activate TLR2/6-dependent NF-κB signalling in a HEK293T reporter cell line compared to wild-type. In addition, the dltD mutant of LGG showed a reduced induction of mRNA of the chemokine IL-8 in the Caco-2 epithelial cell line compared to wild-type. Experiments with highly purified LTA of LGG confirmed that LTA is a crucial factor for IL-8 mRNA induction in Caco-2 epithelial cells. Chemical dealanylation and deacylation reduced IL-8 mRNA expression.
  
  
  CONCLUSIONS
  Taken together, our results indicate that LTA of LGG is a crucial MAMP with pro-inflammatory activities such as IL-8 induction in intestinal epithelial cells and NF-κB induction in HEK293T cells via TLR2/6 interaction. The lipid chains of LGG LTA are needed for these activities, while also the D-alanine substituents are important, especially for IL-8 induction in Caco-2 cells.
authors.affiliation: Centre of Microbial and Plant Genetics, K U Leuven, Kasteelpark Arenberg 20, B-3001, Leuven, Belgium.
authors.forename: Ingmar J J
authors.initials: IJ
authors.lastname: Claes
authors.forename: Marijke E
authors.initials: ME
authors.lastname: Segers
authors.forename: Tine L A
authors.initials: TL
authors.lastname: Verhoeven
authors.forename: Michiel
authors.initials: M
authors.lastname: Dusselier
authors.forename: Bert F
authors.initials: BF
authors.lastname: Sels
authors.forename: Sigrid C J
authors.initials: SC
authors.lastname: De Keersmaecker
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Sarah
authors.initials: S
authors.lastname: Lebeer
mesh_terms: D018938:Caco-2 Cells; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D052201:Lacticaseibacillus rhamnosus / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013682:Teichoic Acids / Q000737:chemistry* / Q000494:pharmacology; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051216:Toll-Like Receptor 6 / Q000378:metabolism
chemical_list: D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D013682:Teichoic Acids; D051195:Toll-Like Receptor 2; D051216:Toll-Like Receptor 6; C009900:lipoteichoic acid