Probiotics are defined as live bacteria which, when administered in sufficient amounts, exert beneficial effects to the gastrointestinal tract. The health benefits of probiotics are dependent on the bacterial strain, and on the clinical setting in which they are used. Indeed, caution needs then to be exerted when administering probiotics in patients with acute inflammation. In fact in patients with severe acute pancreatitis, an increased risk of mortality has been associated with the use of a combination of bacteria employed as probiotic prophylaxis1 and studies carried out on human mucosa organ culture have shown that some probiotics are detrimental in inflammatory bowel diseases.2
The identification of soluble factors produced by probiotics, for which the term of “postbiotics” has recently been coined,2 may represent an opportunity to develop effective new therapeutic strategies that would avoid risks associated with the administration of live bacteria.3 Furthermore, these factors hold the potential for the development of probiotics-fermented functional foods, whose biogenic properties result from the microbial production of bioactive metabolites during the fermentation process.4
Probiotics soluble factors, able of exerting biogenic activities, have been obtained from cell-free culture supernatants of several bacteria cultures. Postbiotics have been identified in several species of Bifidobacteri (breve, lactis, infantis),5–8Bacteroides fragilis,9Escherichia coli Nissle 1917,10–12 and Faecalibacterium prausnitzii.13,14F. prausnitzii is the most abundant bacterium in the human intestinal microbiota of healthy adults, representing >5% of the total bacterial population, and its prevalence is often decreased in conditions of intestinal dysbiosis,15 with low numbers of Faecalibacteria being associated with inflammatory bowel disease.13 This bacterium has anti-inflammatory effects in vitro as well as in vivo, and these effects resulted partly due to its secreted metabolites.14 Nevertheless, evidence of beneficial effects of soluble secreted products by different probiotics strains are progressively increasing, and in recent years there has been an upsurge in research oriented to provide a better understanding of their underlying mechanisms, even if their precise composition is still under investigation. In this regard, most available literature concerns Lactobacilli.
Lactobacillus is a genus of gram-positive facultative anaerobic bacteria (Fig. 1). In nature, there are at least 60 species, constituting the majority of the group of lactic acid bacteria, and so defined for their capacity to convert lactose and other sugars to lactic acid by the lactic fermentation. In humans they are present in the gastrointestinal tract and constitute a small part of the human microbiota. The communication between probiotics and host is multifactorial and involves an integrative repertory of mechanisms that can be categorized as actions that occur within the intestinal lumen, at the mucosal surface, or within and beyond the intestinal mucosa. These include: (1) competition with pathogens for receptor binding, nutrients, and colonization; (2) promotion of intestinal epithelial cell survival and barrier function; and (3) modulation of innate immunity and decrease of pathogen-induced inflammatory bowel diseases.16–18 The beneficial effects of Lactobacilli-secreted products, produced by different species (Table 1), are exerted through mechanisms similar to those described for probiotics.
LACTOBACILLI POSTBIOTIC ACTIVITIES
Firstly, Lactobacilli-secreted factors appear to be a rich source of bacteriocins that restrict the growth and activities of different pathogens5,19–28 (Table 1), potentially lowering the likelihood of infection. One of the first molecules described with potent antipathogenic activity is reuterin, a glycerol derivative compound produced by Lactobacillus reuteri,26 which inhibits a wide spectrum of microorganisms including many bacterial species such as Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Clostridium, Staphylococcus, fungi, and protozoa, many of which are pathogenic for humans. In addition, reuterin, through the prevention of intestinal overgrowth of both commensal and pathogenic microorganisms, plays an important role in the maintenance of a healthy gut microbiota, and shaping and modeling the composition and the spatial architecture of the gastrointestinal microbiota.27
Postbiotics have also been reported to improve mucosal gut barrier integrity through several mechanisms29–34 (Table 1). Lactobacillus rhamnosus GG supernatants ameliorates alcohol-induced increase in intestinal permeability by restoring normal levels of both tight junction proteins and factors involved in mucin production.30 Besides, some supernatants inhibit enterocyte apoptosis and promote growth of intestinal epithelial cells. The effects on cell kinetics appear different according to the intestinal cell type. The soluble molecule, LCrS5-30, isolated from supernatants of a subspecies of Lactobacillus casei (L. casei rhamnosus), promoted human immune cells apoptosis without affecting intestinal epithelial cells.43
Finally, Lactobacilli postbiotics have been reported to modulate inflammatory mediators secretion and NFkB activation reducing tissue inflammation2,11,22,24,27,34–42 (Table 1). Lactobacillus fermentum supernatants have been demonstrated to inhibit the proinflammatory responses of HeLa 229 cells to Yersinia enterocolitica infection,35 through inhibition of IL-8 secretion and decrease of NFkB activation following infection. The protective effects of Lactobacillus paracasei-soluble mediators were firstly observed on dendritic cells cultures exposed to pathogenic Salmonella typhimurium. L. paracasei supernatants were able to inhibit the potential of dendritic cells to both produce inflammatory cytokines (IL-12 and TNF-α) and to drive Th1 T cells in response to Salmonella.36 Using a polarized human mucosal organ culture,2 secretory products of L. paracasei were also shown to affect the capacity of Salmonella to invade and damage the tissue as well as to prevent pathogen-induced proinflammatory cytokines production and decrease in anti-inflammatory cytokine IL10. These effects, associated with a reduction of Salmonella-induced NFkB activation, were also observed on the mucosa of patients with inflammatory bowel disease.2 One of the molecule responsible of these protective effects is likely L. casei-produced/L. paracasei-produced lactocepin which selectively reduced immune cell infiltration and inflammation in experimental inflammatory bowel disease models by the degradation of proinflammatory chemokines.44 Lactocepin, a caseinolytic serine protease, was found to be exclusively present in active fractions of the supernatants.45 The effects of L. rhamnosus supernatants have been firstly observed in young adult mouse colon cells on which it was demonstrated that L. rhamnosus GG supernatants dose-dependently prevented cytokine-induced apoptosis.46 Anti-inflammatory effects of L. rhamnosus GG supernatants have also been observed on primary culture of human colonic smooth muscle cells where supernatants-reversed lipopolysaccharide (LPS) induced NFkB-dependent damage and reduced the LPS-induced morphofunctional cellular alterations.39
LACTOBACILLI FERMENTED FOODS
Fermented functional foods present biogenic properties related to the microbial production of bioactive metabolites produced during the fermentation process.4Lactobacillus helveticus have been used to generate bioactive peptides that exhibit antihypertensive, antimicrobial, and immunomodulatory properties during milk fermentation47,48 and increase osteoblastic bone formation in vitro.49L. paracasei CBA L74-fermented milk preparations for infant formula present a strong anti-inflammatory activity both in vitro, reducing S. typhimurium-induced damage of dendritic cells, and in vivo. In murine dextran sodium sulphate colitis models, these preparations reduce severity of colitis and favor a better recovery.24 These activities were not dependent on the inactivated bacteria but on the metabolic products released during the fermentation process. Finally, L. rhamnosus also has recently been shown to produce exopolysaccharides in skim milk with potential immunomodulatory activity.50 These products suppress cytokines-induced apoptosis and attenuate hydrogen peroxide-induced disruption in intestinal cells in murine models of colitis.40
MECHANISMS OF ACTION
L. rhamnosus GG culture supernatants’ purification resulted in the presence of 2 proteins mostly, one with a molecular mass of around 40 kDa and the other of 75 kDa, namely p40 and p75, respectively. These proteins have also been designated as Msp1 (major secreted proteins 1 or p75) and Msp2 (major secreted proteins 2 or p40).51 Their effects were tested on both several murine and human cell lines and cultured colon explants34 on which it was observed that these 2 proteins prevent TNF-induced intestinal epithelial cells and organ culture damage, inhibit apoptosis, and stimulate proliferative epithelial cells responses. In parallel, the importance of p40 and p75 in mediating these beneficial effects were supported by different lines of evidence.46 First, only Lactobacillus strains that produce p40 and p75 in their supernatant show independence of direct bacterial-cell interaction for these regulatory effects. Second, immunodepletion experiments show that loss of p40 and p75 eliminates L. rhamnosus GG-antiapoptotic effects on the colon epithelial cells.
To date, p40 and p75 are the first identified probiotic bacterial soluble proteins regulating intestinal epithelial homeostasis through specific cellular signal transduction pathways. The antiapoptotic effects of soluble factors are likely associated to a phosphatidylinositol-3′-kinase (PI3K)-dependent Akt activation, subsequent to activation of EGF receptor (EGFR).52 Previous studies have underlined both the importance of EGF in maintaining intestinal health and inducing restitution of the damaged epithelium53 and the crucial regulatory role played by the serine/threonine kinase Akt in different physiological cellular processes (cell differentiation, cell cycle, transcription, translation, metabolism, and apoptosis).54 Akt is able to promote cell survival through a 2-fold way: the inactivation of proapoptotic signals and activation of antiapoptotic signals.55
In particular, on epithelial cells, p40 stimulates the activity of the nonreceptor tyrosine kinase, Src that activates EGFR directly or indirectly through activation of matrix metalloproteinases, which are proteotytic enzymes that induce release of EGFR ligands for transactivation of EGFR and its downstream target, Akt46,56 (Fig. 2A). p40 has also relevant immunoregulatory functions acting on macrophages and lymphocytes to directly downregulate proinflammatory cytokine production.46
On human gastrointestinal colonic smooth muscle, L. rhamnosus GG supernatants resulted in counteracting pathogenic LPS proinflammatory burst and in protecting it from LPS-induced damage. The activity of L. rhamnosus GG supernatant occurred through a direct involvement of TLR2 expressed on human colonic smooth muscle cells.39 Cell surface expression of TLR2 decreased when cells were exposed to supernatants; the absence of available receptors for monoclonal anti-TLR2 binding indirectly substantiating the interaction of LGG with membrane receptors. On cardiomyocites of different species,57,58 TLR2 receptors are associated to the anti-inflammatory PI3K/AKT signaling pathway59 (Fig. 2B).
The available experimental data indicate that probiotic-derived factors, whose presently suggested definition is “postbiotics,” have beneficial properties against pathogen-induced inflammation and related alteration of cytokine release. Several distinct cellular and molecular mechanisms have been proposed and described such the enhancement of the innate immunity, as well as the promotion of intestinal epithelial cell survival and barrier function. The identification of soluble factors mediating the beneficial effects of probiotics may present an opportunity not only to understand their fine mechanisms of action, but also to develop effective pharmacological strategies apted to integrate the action of treatments with live bacteria. Future in vivo studies in animal models will help to determine the feasibility of their use to regulate intestinal inflammatory responses and to promote maintenance of intestinal epithelial integrity.
1. Besselink MG, van Santvoort HC, Buskens E, et al.. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet. 2008; 371:651–659.
2. Tsilingiri K, Barbosa T, Penna G, et al.. Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model. Gut. 2012; 61:1007–1015.
3. Ghishan FK, Kiela PR. From probiotics to therapeutics: another step forward? J Clin Invest. 2011; 121:2149–2152.
4. Stanton C, Ross RP, Fitzgerald GF, et al.. Fermented functional foods based on probiotics and their biogenic metabolites. Curr Opin Biotechnol. 2005; 16:198–203.
5. Muñoz-Quezada S, Bermudez-Brito M, Chenoll E, et al.. Competitive inhibition of three novel bacteria isolated from faeces of breast milk-fed infants against selected enteropathogens. Br J Nutr. 2013; 109:S63–S69.
6. Kondepudi KK, Ambalam P, Nilsson I, et al.. Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile.
Anaerobe. 2012; 18:489–497.
7. Menard S, Candalh C, Bambou JC, et al.. Lactic acid bacteria secrete metabolites retaining anti-inflammatory properties after intestinal transport. Gut. 2004; 53:821–828.
8. Ewaschuk JB, Diaz H, Meddings L, et al.. Secreted bioactive factors from Bifidobacterium infantis
enhance epithelial cell barrier function. Am J Physiol Gastrointest Liver Physiol. 2008; 295:G1025–G1034.
9. Mazmanian SK, Liu CH, Tzianabos AO, et al.. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 2005; 122:107–118.
10. Bär F, Von Koschitzky H, Roblick U, et al.. Cell-free supernatants of Escherichia coli Nissle 1917
modulate human colonic motility: evidence from an in vitro organ bath study. Neurogastroenterol Motil. 2009; 21:559–566.
11. Petrof EO. Probiotics and gastrointestinal diseases: clinical evidence and basic science. Antinflamm Antiallergy Agents Med Chem. 2009; 8:260–269.
12. Prisciandaro LD, Geier MS, Chua AE, et al.. Probiotic factors partially prevent changes to caspases 3 and 7 activation and transepithelial electrical resistance in a model of 5-fluorouracil-induced epithelial cell damage. Support Care Cancer. 2012; 20:3205–3210.
13. Sokol H, Pigneur B, Watterlot L, et al.. Faecalibacterium prausnitzii
is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. PNAS. 2008; 105:16731–16736.
14. Martin R, Chain F, Miguel S, et al.. The commensal bacterium Faecalibacterium prausnitzii
in protective in DNBS-induced chronic moderate and severe colitis models. Inflamm Bowel Dis. 2014; 20:417–430.
15. Miquel S, Martín R, Rossi O, et al.. Faecalibacterium prausnitzii
and human intestinal health. Curr Opin Microbiol. 2013; 16:255–261.
16. Vanderpool C, Yan F, Polk DB. Mechanisms of probiotic action: implications for therapeutic applications in inflammatory bowel diseases. Inflamm Bowel Dis. 2008; 14:1585–1596.
17. Yan F, Polk DB. Probiotics and immune health. Curr Opin Gastroenterol. 2011; 27:496–501.
18. Howarth GS, Wang H. Role of endogenous microbiota, probiotics and their biological products in human health. Nutrients. 2013; 5:58–81.
19. Campana R, Federici S, Ciandrini E, et al.. Antagonistic activity of Lactobacillus acidophilus
ATCC 4356 on the growth and adhesion/invasion characteristics of human Campylobacter jejuni.
Curr Microbiol. 2012; 64:371–378.
20. Ward TL, Hosid S, Ioshikhes I, et al.. Human milk metagenome: a functional capacity analysis. BMC Microbiol. 2013; 13:116–127.
21. Fayol-Messaoudi D, Berger CN, Coconnier-Polter MH, et al.. pH-, lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica
Appl Environ Microbiol. 2005; 71:6008–6013.
22. Chery J, Dvoskin D, Morato FP, et al.. Lactobacillus fermentum
, a pathogen in documented cholecystitis. Int J Sur Case Rep. 2013; 4:662–664.
23. Pridmore RD, Pittet AC, Praplan F, et al.. Hydrogen peroxide production by Lactobacillus johnsonii
NCC 533 and its role in anti-Salmonella
activity. FEMS Microbiol Lett. 2008; 283:210–215.
24. Zagato E, Mileti E, Massimiliano L, et al.. Lactobacillus paracasei
CBA L74 metabolic products and fermented milk for infant formula have anti-inflammatory activity on dendritic cells in vitro and protective effects against colitis and an enteric pathogen in vivo. PLoS One. 2014; 9:e87615.
25. Muller DM, Carrasco MS, Tonarelli GG, et al.. Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum
Lp 31 strain isolated from dry-fermented sausage. J Appl Microbiol. 2009; 106:2031–2040.
26. Talarico TL, Dobrogosz WJ. Chemical characterization of an antimicrobial substance produced by Lactobacillus reuteri.
Antimicrob Agents Chemother. 1989; 33:674–679.
27. Jones SE, Versalovic J. Probiotic Lactobacillus reuteri
biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiol. 2009; 9:35–48.
28. Gomes BC, Rodrigues MR, Winkelstroter LK, et al.. In vitro
evaluation of the probiotic potential of bacteriocin producer Lactobacillus sakei.
J Food Prot. 2012; 75:1083–1089.
29. Wang Y, Kirpich I, Liu Y, et al.. Lactobacillus rhamnosus
GG treatment potentiates intestinal hypoxia-inducible factor, promotes intestinal integrity and ameliorates alcohol-induced liver injury. Am J Pathol. 2011; 179:2866–2875.
30. Wang Y, Liu Y, Sidhu A, et al.. Lactobacillus rhamnosus
GG culture supernatant ameliorates acute alcohol-induced intestinal permeability and liver injury. Am J Physiol Gastrointest Liver Physiol. 2012; 303:G32–G41.
31. Lin PW, Nasr TR, Berardinelli AJ, et al.. The probiotic Lactobacillus
GG may augment intestinal host defense by regulating apoptosis and promoting cytoprotective responses in the developing murine gut. Pediatr Res. 2008; 64:511–516.
32. Yan F, Polk DB. Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells. J Biol Chem. 2002; 277:50959–50965.
33. Yan F, Liu L, Dempsey PJ, et al.. A Lactobacillus rhamnosus
GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor. J Biol Chem. 2013; 288:30742–30751.
34. Yan F, Cao H, Cover TL, et al.. Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Gastroenterology. 2007; 132:562–575.
35. Frick JS, Schenk K, Quitadamo M, et al.. Lactobacillus fermentum
attenuates the proinflammatory effect of Yersinia enterocolitica
on human epithelial cells. Inflamm Bowel Dis. 2007; 13:83–90.
36. Bermudez-Brito M, Muñoz-Quezada S, Gomez-Llorente C, et al.. Human intestinal dendritic cells decrease cytokine release against Salmonella
infection in the presence of Lactobacillus paracasei
upon TLR activation. PLoS One. 2012; 7:e43197.
37. Yan F, Cao H, Cover TL, et al.. Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism. J Clin Invest. 2011; 121:2242–2253.
38. Wang Y, Liu Y, Kirpich I, et al.. Lactobacillus rhamnosus
GG reduces hepatic TNFα production and inflammation in chronic alcohol-induced liver injury. J Nutr Biochem. 2013; 24:1609–1615.
39. Ammoscato F, Scirocco A, Altomare A, et al.. Lactobacillus rhamnosus
protects human colonic muscle from pathogen lipopolysaccharide-induced damage. Neurogastroenterol Motil. 2013; 25:984–e777.
40. Yoda K, Miyazawa K, Hosoda M, et al.. Lactobacillus
GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor. Eur J Nutr. 2014; 53:105–115.
41. Paszti-Gere E, Szeker K, Csibrik-Nemeth E, et al.. Metabolites of Lactobacillus plantarum
2142 prevent oxidative stress-induced overexpression of proinflammatory cytokines in IPEC-J2 cell line. Inflammation. 2012; 35:1487–1499.
42. Nemeth E, Fajdiga S, Malago J, et al.. Inhibition of Salmonella
-induced IL-8 synthesis and expression of Hsp70 in enterocyte-like Caco-2 cells after exposure to non-starter Lactobacilli. Int J Food Microbiol. 2006; 112:266–274.
43. Chiu Yi-Han, Hsieh Yi-Jen, Liao Kuang-Wen, et al.. Preferential promotion of apoptosis of monocytes by Lactobacillus casei rhamnosus
soluble factors. Clin Nutr. 2010; 29:131–140.
44. Hörmannsperger G, von Schillde MA, Haller D. Lactocepin as a protective microbial structure in the context of IBD. Gut Microbes. 2013; 4:152–157.
45. Von Shillde MA, Hörmannsperger G, Weiher M, et al.. Lactocepin secreted by Lactobacillus
exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines. Cell Host Microbe. 2012; 11:387–396.
46. Yan F, Polk DB. Characterization of a probiotic-derived soluble protein which reveals a mechanism of preventive and treatment effects of probiotics on intestinal inflammatory diseases. Gut Microbes. 2012; 3:25–28.
47. LeBlanc JG, Matar C, Valdez JC, et al.. Immunomodulating effects of peptidic fractions issued from milk fermented with Lactobacillus helveticus.
J Dairy Sci. 2002; 85:2733–2742.
48. Seppo L, Jauhiainen T, Poussa T, et al.. A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am J Clin Nutr. 2003; 77:326–330.
49. Narva M, Halleen J, Vaananen K, et al.. Effects of Lactobacillus helveticus
fermented milk on bone cells in vitro. Life Sci. 2004; 75:1727–1734.
50. Shao L, Wu Z, Zhang H, et al.. Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus
KF5. Carbohydr Polym. 2014; 107:51–56.
51. Claes IJJ, Schoofs G, Regulski K, et al.. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted proteins of Lactobacillus rhamnosus
GG. PLoS One. 2012; 7:e31588.
52. Iyer C, Kosters A, Sethi G, et al.. Probiotic Lactobacillus reuteri
promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kappaB and MAPK signalling. Cell Microbiol. 2008; 10:1442–1452.
53. Franke TF, Yang SI, Chan TO, et al.. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995; 81:727–736.
54. Thompson JE, Thompson CB. Putting the rap on Akt. J Clin Oncol. 2004; 22:4217–4226.
55. Aggarwal BB. Nuclear factor-kB: the enemy within. Cancer Cell. 2004; 6:203–208.
56. Gschwind A, Zwick E, Prenzel N, et al.. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene. 2001; 20:1594–1600.
57. Ha T, Hu Y, Liu L, et al.. TLR2 ligands attenuate cardiac dysfunction in polymicrobial sepsis via a phosphoinositide 3-kinase-dependent mechanism. Am J Physiol Heart Circ Physiol. 2010; 298:H984–H991.
58. Hu W, Li F, Mahavadi S, et al.. Upregulation of RGS4 expression by IL-1beta in colonic smooth muscle is enhanced by ERK1/2 and p38 MAPK and inhibited by the PI3K/Akt/GSK3beta pathway. Am J Physiol Cell Physiol. 2009; 296:1310–1320.
59. Fukao T, Koyasu S. PI3K and negative regulation of TLR signaling. Trends Immunol. 2003; 24:358–363.