skip to content »

Pathology

Houston, Texas

Versalovic Lab
Pathology
not shown on screen

General Concepts of Probiotics

1. Rationale for Studies of Probiotic Lactobacillus Species

The rational selection of probiotic bacteria provides opportunities for the prevention and treatment of IBD, based on recent evidence from animal and human studies (1,2). "Old friends" or beneficial microbes such as Lactobacillus strains may regulate immune responses directly in the host (3,4). Selective deficiencies of intestinal lactobacilli and bifidobacteria have been described in patients with Crohn’s disease (CD) (5). Supplementation with probiotic Lactobacillus species has been effective at ameliorating intestinal inflammation in rodent colitis models and human patients with IBD. Combination probiotic therapy reduced the severity of recurrent Th1-mediated murine colitis by induction of IL-10-producing regulatory T lymphocytes (6). Administration of Lactobacillus rhamnosus (LGG) to children with Crohn’s disease resulted in significant reductions of the Crohn’s disease activity index (CDAI) four weeks after initiation of therapy (7). Probiotic formulations that included four different Lactobacillus species have been effective for the prevention (8) or treatment (9) of IBD-related pouchitis. Combination probiotic therapy induced remission in a majority of patients with active ulcerative colitis not responding to conventional therapy, and probiotic organisms were detected in biopsy samples from responders (10). In contrast to E. coli and Lactobacillus crispatus, specific Lactobacillus bulgaricus and Lactobacillus casei clones significantly reduced pro-inflammatory cytokine responses ex vivo in human intestinal tissue explants obtained from patients with Crohn’s disease (11,12). These results suggest that defined probiotic Lactobacillus clones may reduce inflammation in the intestinal mucosa of patients with IBD by direct immunosuppression. The modification of microbial ecology in the human intestine and introduction of specific probiotic bacteria into a complex microbiota may prevent recurrence or diminish inflammation in human IBD (13).

The genus Lactobacillus includes greater than 100 different species, and has received attention as a possible source of probiotic agents and protein delivery systems for the mammalian intestine (14-16). To date, fewer than 20 species have been found consistently in the mammalian gastrointestinal tract. In the mouse, a subset of Lactobacillus species have been characterized in the gastrointestinal tracts of healthy animals (17,18). Only a restricted subset of Lactobacillus species has defined probiotic activity in animal and human studies. Several Lactobacillus species stably colonize and persist in the mammalian intestine (19-21). Bacteria of the Lactobacillus/Enterococcus group colonize the crypts and the interlaced layer (includes mucus layer and epithelial surface) adjacent to the mucosa in the murine large intestine (22).

2. Why Study Lactobacillus reuteri and Lactobacillus paracasei in Particular?

L. reuteri is used in commercial probiotic formulations and is the most widely distributed Lactobacillus species among animals (23). L. reuteri is a predominant Lactobacillus species in the mouse intestine and has been isolated from the small and large intestine (17,18). Lactobacillus reuteri is considered to be one of a limited number of indigenous Lactobacillus species in the human gastrointestinal tract (24). Interestingly, L. reuteri is the only Lactobacillus species currently considered as an indigenous species in both mouse and human intestinal tracts. L. reuteri demonstrated the ability to protect human infants from gastrointestinal infections (25) and reduced sick leave in the workplace among healthy adults (26). L. paracasei conferred probiotic effects in the mouse intestine by suppressing humoral and cellular immune responses (27) and gut muscle hypercontractility in a post-infectious irritable bowel syndrome model (28). L. paracasei stimulated IL-10 production by peptidase-mediated degradation of mammalian lactoglobulin (29).

Figure 1. L. reuteri Colonizes the Human Intestinal Ileum
Figure 1. L. reuteri Colonizes the Human Intestinal Ileum

L. reuteri has demonstrated probiotic activity in the human and mouse gastrointestinal tracts (23), and viable L. reuteri is essential for immunomodulatory effects on human cells (30). Although bacterial DNA may confer probiotic effects in the mouse intestine (31), other studies support the idea that viable probiotic bacteria are important for persistent and stable effects in the host (20,32). L. reuteri colonization has been demonstrated in the human duodenum and ileum by FISH of biopsy specimens (21) (Fig. 1). The molecular beacon probe targets a species-specific sequence in L. reuteri and demonstrates Lactobacillus reuteri colonization (apple-green fluorescence) adjacent to human intestinal mucosa by FISH (Fig. 1). Administration of viable L. reuteri to human volunteers resulted in stable colonization for at least 28 days in the small intestine and modulation of intestinal immune responses (21). Recent data indicate that viable L. reuteri organisms were required for anti-inflammatory effects in intestinal epithelial cell models (30). L. reuteri organisms have potent immunosuppressive activities when mixed with murine dendritic cells (33). L. reuteri treatment of rotaviral gastfroenteritis in human patients reduced the duration of disease and facilitated patient recovery (34,35). In the acetic acid-induced rat colitis model, L. reuteri yielded superior beneficial effects when compared with Lactobacillus rhamnosus and diminished mucosal inflammation in the intestine (36). L. reuteri has been isolated from the mouse jejunum, cecum, and colon in mice lacking evidence of colitis (17). Specific L. reuteri genes are induced in the mouse gastrointestinal tract and may encode factors that modulate intestinal function (37).

3. The Importance of Studying Defined Clones of Probiotic Lactobacillus

Probiosis, like microbial pathogenesis, ultimately has a clonal basis and depends on the functional characterization of specific bacterial strains. Individual bacterial clones have stable genetic features that confer probiotic functions within specific bacterial lineages. Lactobacillus species differed in their ability to modulate pro-inflammatory cytokine production in bone marrow-derived dendritic cells, and cytokine-inhibitory L. reuteri antagonized effects of cytokine-inducing L. casei (33). L. reuteri and L. plantarum clones demonstrated differential inhibition of pro-inflammatory cytokine production with L. reuteri showing the most potent immunosuppressive effects (33). Investigators have described the up-regulation of pro-inflammatory cytokine responses and NF-?B activation by intact lactobacilli (38-40). Cytokine modulatory effects are strain-dependent (18,33) and vary with growth phase and cell preparation methods (33,38-41).

4. Intestinal Bacteria May Directly Suppress Innate Immune Responses

The innate immune system represents the primary interface between the microbial world and the host (42,43). The molecular bases of interactions between bacteria and innate immunity may hold the key to a greater understanding of probiotic immunosuppressive mechanisms of action. A variety of bacteria directly modulate cytokine networks and innate immune responses by multiple mechanisms (44). Messenger molecules that are important for intercellular communication among bacteria (e.g. quorum sensing) may affect host intestinal physiology (45). The quorum sensing molecule, 3-oxododecanoyl-L-homoserine lactone, produced by Pseudomonas aeruginosa suppressed the production of interleukin-12 and TNF-α by lipopolysaccharide-stimulated macrophages (46). Intestinal bacteria may diminish pro-inflammatory cytokine production by inhibiting NF-κB activation. Avirulent Salmonella inhibited the ubiquitination of the NF-κB inhibitory subunit, IκB-α (47), and Bacteroides thetaiotamicron modulated the NF-κB signaling pathway by facilitating the nuclear export of RelA via PPAR-λ (48). The intestinal bacterium Yersinia enterocolitica expressed a protein YopP that interferes with TNF-α production in murine macrophages by interfering with the NF-κB and MAPK pathways (49). Microbial networks and the balance between pro-inflammatory and anti-inflammatory components of the intestinal microbiota represent a poorly understood part of the intestinal landscape.

5. Probiotic Lactobacillus Directly Suppress Inflammation

Figure 2. Specific Lactobacillus Clones Suppress Human TNF Production in Crohn’s MucosaCommensal bacteria produce immunoregulatory factors, or "immunomodulins" that directly suppress cytokine production (44). Different Lactobacillus species, including L. reuteri, inhibited pro-inflammatory cytokine production in bone marrow-derived dendritic cells (33). In contrast, IL-10 production by dendritic cells was maintained at similar levels. Different Lactobacillus strains may differ with respect to immunosuppressive effects. In contrast to Lactobacillus crispatus, Lactobacillus casei and Lactobacillus bulgaricus strains down-regulated TNF-α production by human intestinal explants from patients with Crohn’s disease (Fig. 2) (11,12). Viable bacteria were added to organ cultures of human ileal explants, and only L. casei and L. bulgaricus strains significantly inhibited intestinal TNF-α secretion in inflamed tissue (black bars) and non-inflamed tissue (white bars) (** p<0.01) (Fig. 2) (11). The inhibition of cytokine production may result from the secretion of soluble mediators by probiotic bacteria. Lactic acid bacteria secreted anti-inflammatory metabolites (<10 kDa) that inhibited human TNF-a production by human peripheral blood mononuclear cells (50). In addition to direct effects on cytokine production, probiotic bacteria may stimulate regulatory T cell responses and corresponding regulatory cytokine production. L. paracasei induced the expansion of regulatory CD4+ T cell populations which produced high levels of regulatory cytokines such as IL-10 and transforming growth factor-β (TGF-β) (51).

References

  1. Sartor RB. Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: Antibiotics, probiotics, and prebiotics. Gastroenterology 2004;126(6):1620-33.
  2. Fedorak RN and Madsen KL. Probiotics and the management of inflammatory bowel disease. Inflamm Bowel Dis 2004;10(3):286-99.
  3. Rook GA, Adams V, Hunt J, Palmer R, Martinelli R and Brunet LR. Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders. Springer Semin Immunopathol 2004;25(3-4):237-55.
  4. Guarner F, Bourdet-Sicard R, Brandtzaeg P, Gill HS, McGuirk P, van Eden W, Versalovic J, Weinstock JV and Rook GA. Mechanisms of disease: The hygiene hypothesis revisited. Nat Clin Pract Gastroenterol Hepatol 2006;3(5):275-84.
  5. Giaffer MH, Holdsworth CD and Duerden BI. The assessment of faecal flora in patients with inflammatory bowel disease by a simplified bacteriological technique. J Med Microbiol 1991;35(4):238-43.
  6. Di Giacinto C, Marinaro M, Sanchez M, Strober W and Boirivant M. Probiotics ameliorate recurrent th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-ß-bearing regulatory cells. J Immunol 2005;174(6):3237-46.
  7. Gupta P, Andrew H, Kirschner BS and Guandalini S. Is Lactobacillus GG helpful in children with Crohn's disease? Results of a preliminary, open-label study. J Pediatr Gastroenterol Nutr 2000;31(4):453-7.
  8. Gionchetti P, Rizzello F, Helwig U, Venturi A, Lammers KM, Brigidi P, Vitali B, Poggioli G, Miglioli M and Campieri M. Prophylaxis of pouchitis onset with probiotic therapy: A double-blind, placebo-controlled trial. Gastroenterology 2003;124(5):1202-9.
  9. Gionchetti P, Rizzello F, Venturi A, Brigidi P, Matteuzzi D, Bazzocchi G, Poggioli G, Miglioli M and Campieri M. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: A double-blind, placebo-controlled trial. Gastroenterology 2000;119(2):305-9.
  10. Bibiloni R, Fedorak RN, Tannock GW, Madsen KL, Gionchetti P, Campieri M, De Simone C and Sartor RB. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am J Gastroenterol 2005;100(7):1539-46.
  11. Borruel N, Carol M, Casellas F, Antolin M, de Lara F, Espin E, Naval J, Guarner F and Malagelada JR. Increased mucosal tumour necrosis factor alpha production in Crohn's disease can be downregulated ex vivo by probiotic bacteria. Gut 2002;51(5):659-64.
  12. Borruel N, Casellas F, Antolin M, Llopis M, Carol M, Espiin E, Naval J, Guarner F and Malagelada JR. Effects of nonpathogenic bacteria on cytokine secretion by human intestinal mucosa. Am J Gastroenterol 2003;98(4):865-70.
  13. Rastall RA, Gibson GR, Gill HS, Guarner F, Klaenhammer TR, Pot B, Reid G, Rowland IR and Sanders ME. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: An overview of enabling science and potential applications. FEMS Microbiol Ecol 2005;52(2):145-52.
  14. Tannock GW. Probiotic properties of lactic-acid bacteria: Plenty of scope for fundamental R & D. Trends Biotechnol 1997;15(7):270-4.
  15. Seegers JF. Lactobacilli as live vaccine delivery vectors: Progress and prospects. Trends Biotechnol 2002;20(12):508-15.
  16. Neu T and Henrich B. New thermosensitive delivery vector and its use to enable nisin-controlled gene expression in Lactobacillus gasseri. Appl Environ Microbiol 2003;69(3):1377-82.
  17. Madsen KL, Doyle JS, Jewell LD, Tavernini MM and Fedorak RN. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 1999;116(5):1107-14.
  18. Peña JA, Li SY, Wilson PH, Thibodeau SA, Szary AJ and Versalovic J. Genotypic and phenotypic studies of murine intestinal lactobacilli: Species differences in mice with and without colitis. Appl Environ Microbiol 2004;70(1):558-68.
  19. de Champs C, Maroncle N, Balestrino D, Rich C and Forestier C. Persistence of colonization of intestinal mucosa by a probiotic strain, Lactobacillus casei subsp. rhamnosus LCR35, after oral consumption. J Clin Microbiol 2003;41(3):1270-3.
  20. McCarthy J, O'Mahony L, O'Callaghan L, Sheil B, Vaughan EE, Fitzsimons N, Fitzgibbon J, O'Sullivan GC, Kiely B, Collins JK and Shanahan F. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 2003;52(7):975-80.
  21. Valeur N, Engel P, Carbajal N, Connolly E and Ladefoged K. Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract. Appl Environ Microbiol 2004;70(2):1176-81.
  22. Swidsinski A, Loening-Baucke V, Lochs H and Hale LP. Spatial organization of bacterial flora in normal and inflamed intestine: A fluorescence in situ hybridization study in mice. World J Gastroenterol 2005;11(8):1131-40.
  23. Casas IA and Dobrogosz WJ. Validation of the probiotic concept: Lactobacillus reuteri confers broad-spectrum protection against disease in humans and animals. Microbial Ecol in Health Dis 2000;12(247-85).
  24. Reuter G. The Lactobacillus and Bifidobacterium microflora of the human intestine: Composition and succession. Curr Issues Intest Microbiol 2001;2(2):43-53.
  25. Weizman Z, Asli G and Alsheikh A. Effect of a probiotic infant formula on infections in child care centers: Comparison of two probiotic agents. Pediatrics 2005;115(1):5-9.
  26. Tubelius P, Stan V and Zachrisson A. Increasing work-place healthiness with the probiotic Lactobacillus reuteri: A randomised, double-blind placebo-controlled study. Environ Health 2005;4(25).
  27. Prioult G, Fliss I and Pecquet S. Effect of probiotic bacteria on induction and maintenance of oral tolerance to beta-lactoglobulin in gnotobiotic mice. Clin Diagn Lab Immunol 2003;10(5):787-92.
  28. Verdu EF, Bercik P, Bergonzelli GE, Huang XX, Blennerhasset P, Rochat F, Fiaux M, Mansourian R, Corthesy-Theulaz I and Collins SM. Lactobacillus paracasei normalizes muscle hypercontractility in a murine model of postinfective gut dysfunction. Gastroenterology 2004;127(3):826-37.
  29. Prioult G, Pecquet S and Fliss I. Stimulation of interleukin-10 production by acidic beta-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei NCC2461 peptidases. Clin Diagn Lab Immunol 2004;11(2):266-71.
  30. Ma D, Forsythe P and Bienenstock J. Live Lactobacillus reuteri is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression. Infect Immun 2004;72(9):5308-14.
  31. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky B, Akira S, Takeda K, Lee J, Takabayashi K and Raz E. Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology 2004;126(2):520-8.
  32. Galdeano CM and Perdigon G. Role of viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation. J Appl Microbiol 2004;97(4):673-81.
  33. Christensen HR, Frokiaer H and Pestka JJ. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J Immunol 2002;168(1):171-8.
  34. Pearce JL and Hamilton JR. Controlled trial of orally administered lactobacilli in acute infantile diarrhea. J Pediatr 1974;84(2):261-2.
  35. Shornikova AV, Casas IA, Mykkanen H, Salo E and Vesikari T. Bacteriotherapy with Lactobacillus reuteri in rotavirus gastroenteritis. Pediatr Infect Dis J 1997;16(12):1103-7.
  36. Holma R, Salmenpera P, Lohi J, Vapaatalo H and Korpela R. Effects of Lactobacillus rhamnosus GG and Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats. Scand J Gastroenterol 2001;36(6):630-5.
  37. Walter J, Heng NC, Hammes WP, Loach DM, Tannock GW and Hertel C. Identification of lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Appl Environ Microbiol 2003;69(4):2044-51.
  38. Miettinen M, Vuopio-Varkila J and Varkila K. Production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria. Infect Immun 1996;64(12):5403-5.
  39. Miettinen M, Matikainen S, Vuopio-Varkila J, Pirhonen J, Varkila K, Kurimoto M and Julkunen I. Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells. Infect Immun 1998;66(12):6058-62.
  40. Miettinen M, Lehtonen A, Julkunen I and Matikainen S. Lactobacilli and streptococci activate NF-kappa B and STAT signaling pathways in human macrophages. J Immunol 2000;164(7):3733-40.
  41. Peña JA and Versalovic J. Lactobacillus rhamnosus GG decreases TNF-a production in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism. Cell Microbiol 2003;5(4):277-85.
  42. Aderem A and Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406(6797):782-7.
  43. Janeway CA, Jr. and Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20(197-216).
  44. Wilson M, Seymour R and Henderson B. Bacterial perturbation of cytokine networks. Infect Immun 1998;66(6):2401-9.
  45. Sperandio V, Torres AG, Jarvis B, Nataro JP and Kaper JB. Bacteria-host communication: The language of hormones. Proc Natl Acad Sci U S A 2003;100(15):8951-6.
  46. Telford G, Wheeler D, Williams P, Tomkins PT, Appleby P, Sewell H, Stewart GS, Bycroft BW and Pritchard DI. The Pseudomonas aeruginosa quorum-sensing signal molecule n-(3-oxododecanoyl)-l-homoserine lactone has immunomodulatory activity. Infect Immun 1998;66(1):36-42.
  47. Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS and Madara JL. Prokaryotic regulation of epithelial responses by inhibition of IKB-a ubiquitination. Science 2000;289(5484):1560-3.
  48. Kelly D, Campbell JI, King TP, Grant G, Jansson EA, Coutts AG, Pettersson S and Conway S. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of ppar-gamma and rela. Nat Immunol 2004;5(1):104-12.
  49. Boland A and Cornelis GR. Role of YopP in suppression of tumor necrosis factor alpha release by macrophages during yersinia infection. Infect Immun 1998;66(5):1878-84.
  50. Menard S, Candalh C, Bambou JC, Terpend K, Cerf-Bensussan N and Heyman M. Lactic acid bacteria secrete metabolites retaining anti-inflammatory properties after intestinal transport. Gut 2004;53(6):821-8.
  51. von der Weid T, Bulliard C and Schiffrin EJ. Induction by a lactic acid bacterium of a population of CD4(+) T cells with low proliferative capacity that produce transforming growth factor beta and interleukin-10. Clin Diagn Lab Immunol 2001;8(4):695-701.