Probiotic bacteria: mechanisms of action?
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1 Probiotic bacteria: mechanisms of action? Probiotic derived antibacterial substances (microcins) Competitive inhibition of pathogens Modulation of inflammatory responses: Increasing antiinflammatory cytokine production Suppressing proinflammatory cytokine production Modulation of TLR s Induction of barrier effector molecules
2 The mucus barrier: defensins + mucins Normal Few microbes Intestinal Lumen (an)aerobic bacteria Outer mucus layer (700 um) mucins (-) and antibacterial peptides (+) (defensins, cathelicidines) Crohn s disease Sterile Inner mucus layer (100 um) adherent, rich in antibacterial peptides Mucosa (200 um) epithelial cell barrier secretion of mucus and defensins etc. A. Swidsinski
3 A Plethora of Antibacterial Peptides Constitutive HBD-1 alpha and beta-defensins Cathelicidins Elafin / Antiproteases BPI Lysozyme Permanent war at host interface
4 Binding of antimicrobial peptides to mucins in mucus while retaining functional activity Mucus (mucins) binding HBD-2 Staining with SYTO 9 (living bacteria green) and Propidiumiodide (dead bacteria red) E. coli ATCC Untreated control E. coli ATCC HBD-2 Incubated with colonic mucus Dr. Sabine Nuding
5 Probiotic E. coli in IBD: Background First Case Report on the Treatment of Colitis with E. coli Nissle 1917 (MUTAFLOR ) A. Nissle (1918). Die antagonistische Behandlung chronischer Darmstörungen mit Colibakterien. Med. Klinik No 2: Mon 12 Mon 12 Mon Mesalazin E. coli Alfred Nissle 20 0 Kruis I Rembacken Kruis II Treatment with the non pathogenic E. coli Nissle is equivalent to mesalazine in maintaining remission in ulcerative colitis Rembacken et al., Lancet 1999 Kruis et al., Alimentary Pharmacol Ther 1997 Kruis et al., Gut 2005 Current consensus: Probiotic E. coli Nissle (and other Probiotic strains) seem to be effective in maintaining remission of ulcerative colitis, rather no effect in Crohn s disease
6 Defensin expression in IBD Defensins = antimicrobial peptides Spectrum: gram-positive and gram-negative bacteria, viruses Lack of induction of the human beta defensin-2 (hbd-2) in inflamed Crohn s disease and in noninflamed UC Hypothesis: The benefit from E. coli Nissle in maintenance of remission may be due to induction of defensins Wehkamp et al, IBD 2003:
7 relative transcript level HBD-2 Induction of the human β-defensin-2 in intestinal epithelial cells by E. coli Nissle 25 ** * unstimulated E. coli Nissle 1917 * p < 0,001 ** p < 0,0001 EPEC K12 DSM498 UPEC PZ 865 PZ 860 PZ 862 PZ 866 PZ 915 E. coli strains * p < 0,001 ** p < 0,0001 PZ 861 PZ 864 PZ 868 PZ 870 PZ 863 PZ 867 PZ 835 PZ 830 EcN K12 Unst. 30 more E. coli isolates tested: all negative. E. coli Nissle is quite unique among E. coli s Wehkamp et al. Infection and Immunity 2004
8 Mechanism?
9 The relative importance of the NF-κB and AP-1 binding sites in the hbd-2 promoter NF- B NF- B AP-1 +1 hbd luc Exon 1-572/ / /-127 NF- B NF- B AP-1 +1 NF- B-mut1-luc Exon 1-572/ / /-127 NF- B NF- B AP-1 +1 NF- B-mut2-luc Exon 1-572/ / /-127 NF- B NF- B AP-1 +1 NF- B-mut1+2-luc Exon 1-572/ / /-127 NF- B NF- B AP-1 +1 AP-1-mut-luc Exon 1-572/ / /-127 NF- B NF- B AP-1 +1 AP-1+NF- B-mut-luc Exon 1-572/ / /-127 Fold increase NF- B-mut1+2+AP-1-luc AP-1-mut-luc control Nissle 1917 NF- B-mut1+2-luc NF- B-mut2-luc NF- B-mut1-luc hbd luc Relative hbd-2 promoter activity Probiotic induction of defensins (HBD2) is NF-κB and AP-1 dependent Wehkamp et al. Infection and Immunity 2004
10 Characteristics of E. coli Nissle 1917 Iron acquisition systems Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Small cryptic plasmids 3 kb 5 kb chu aer cbt ent ybt cit mch chromosome STOP rfb mcm kps Microcins fla fim foc csg Fimbriae Flagellae of H1 type Special LPS of O6 type K5 capsule O6 : K5 : H1 serotype
11 Which component of E. coli Nissle is the effector? p<0.01 Special E. coli Nissle Flagellin! Schlee et al. Infection and Immunity 2007
12 hbd-2 mrna induction in % Induction of defensins: what about other probiotics? E. coli Nissle 1917 K12 L. gasseri PZ 1160 L. ac. PZ 1030 L. ac. PZ 1129 L. ac. PZ 1130 L. ferm. PZ 1162 Leuconostoc * * * * L. paracasei L. plantarum Pediococcus Cocktail Wehkamp et al. Infection and Immunity 2004 Schlee et al. Clin and Exp. Immun 2008 The effect is not specifc to E. coli Nissle but can be found in other probiotic bacteria
13 Fecal HBD-2 peptide concentration in volunteers HBD-2 expression in cell culture a 1 in vivo a 2 in vitro b b c E. Coli probiotic (Symbioflor) treatment induces intestinal secretion of HBD-2 peptide in man
14 Antimicrobial Activity against Probiotics Suicidal effect of probiotic stimulation? Explanation for need of daily delivery?
15 Ulcerative colitis? is associated with thinner mucus layer Pullan et. al. Gut 1994 mucus depletion is associated with intestinal inflammation Johannson et al PNAS 2008 ahead of print
16 Some probiotics affect the barrier Inducible defensin formation Mucin (Muc 1) synthesis The combination of both
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