Medical Hypothesis

Mechanism of Probiotic Exopolysaccharide in Immune

stimulation and Immune tolerance through TGF B pathway

Edjohn Aaron Macauyag

Institute of Biology, University of the Philippines Diliman

ABSTRACT

The proposal of this paper is that Probiotics cause either immunostimulation

or immune tolerance havingis strain dependent properties and is made
through receptor recognition of the EPS type and structure. This bipolar role
is proposed to be made possible through the induction of the TGF B pathway
by the EPS which in turn stimulates the FOXP3+ regulatory T cells that is
involved in immune homeostasis as it inhibits cell activation and differentiation
that leads to immune suppression; while it also stimulates immune stimulation by
converting immature T cells into iTregs and Th17 to strengthen the fight against
pathogens. Two models are proposed Model 1 having the presence of single EPS

receptor with co stimulant for immune stimulation and inhibition and Model 2
having different EPS receptors for immune inhibition and stimulation with an
unknown sorter.

INTRODUCTION

In the maintenance of health, a critical balance should be maintained between

protective mucosal immunity, like defense against pathogens and systemic
hyporesponsiveness or immune tolerance non harmful antigens like food. Lactic

acid bacteria especially Lactobacilli are known to have immunomodulation

effects and recently are reported to be involved in tolerance for
hypersensitivity. Studies in vitro and in vivo in mice showed
immunomodulation by local increased levels of igA and cytokine levels in the
presence of LAB and even just the exopolysaccharide of LAB. [1] This is
supported by another study on EPS showing lower than LPS immune
stimulation cytokine by production in macrophages, specifically tumor
necrosis factor (TNF-), IL-12 and interleukin 6 (IL-6. In addition, they also
reported hyporesponsiveness, through reduced production of TNF- after
restimulation with either LPS or EPS and showed cross-tolerance. This is in
line with other studies showing immune regulation effects of LAB EPS [3,4]
These studies altogether suggest a complexity in immune function of EPS
and prompts a more thorough study of EPS structure and composition and its
relation to immune responses. In line with this there has been a hypothesis
trying to relate the physicochemical properties of EPS with their immune
modulation effects saying that EPS having negative charge and/or small
molecular weight can have mild stimulatory effects on immune cells, while
uncharged polymers and have high molecular weight have suppressive
capabilities. [2] However, studies show EPS immune effects are strain-

dependent and produce both pro- and anti-inflammatory cytokines various

ways [5]. This suggests a more complex reaction and signal processing.
The bridge between microbiota and the immune system is the mucosaassociated lymphoid tissue (MALT). MALT consists about half the lymphocytes the
immune system has [8] and is located along the surfaces of all tissues of the
mucosal linings of organs. MALT consists of highly known subgroups such as gutassociated lymphoid tissue (GALT), and bronchus-associated lymphoid tissue (BALT)
nasopharynx-associated lymphoid tissue (NALT) and lesser known subgroups like
conjunctiva-associated lymphoid tissue (CALT), larynx-associated (LALT) lacrimal
duct-associated (LDALT), and salivary duct-associated lymphoid tissue (DALT).[6]
MALT has the main function to produce IgA at mucosal surfaces that are antigen
specific and Th2-dependent but could also result in immune tolerance by inducing
Th1 and cytotoxic T-cell mediated depending on the stimuli received [7] .

Therefore further studies on EPS structure on different strains, costimulation

with other bacterial surface compounds and immune cell receptor studies are
needed to further elucidate the mechanism involved in these processes. In
this paper, the bipolar function of probiotics is proposed to be caused by
receptor mediated interactions with probiotic EPS and is transduced using
the Transforming growth factor (TGF) beta1 pathway by stimulation of FOXP3+
regulatory T cells.

HYPOTHESIS

Probiotic bacteria play a dual role in immune function:The increased gutimmune response against pathogens and promotion of oral tolerance for
harmless antigens. This polarized regulation of the immune response to
gastrointestinal antigens is consistent among various studies[1,3,7,8] there
are many mechanisms by which Probiotics could affect immune function. Non
antigen specific indirect effects are: reinforcement of hyperpermeable
epithelial-mucosal barriers [13,14], increased production of short chain fatty
acids [15] and synthesis of antimicrobial peptides[16].On the other hand, strain
specific direct effects are also produced such as acting as ligands for innate immune
system receptors like Toll-like receptors and nucleotide-binding oligomerization
domain receptors, [18,19]; influencing development and differentiation of
immune cells like dendritic cells and T cells [17]synthesis of the important

regulatory cytokines IL-10 and TGF-B [17, 20]. These direct and strain
specific stimulation is proposed in this paper to be made possible by
diversity in EPS structure, in different strains sorted by immune cell receptors
and the bipolar action could possibly be attributed to the stimulation and
dual effect of cytokine transforming growth factor [10] . TGF beta exists in
three isoforms 1, 2, and 3 with TGF-1 being most prevalent type and displays a
broad spectrum of activities in mucosal immune regulation, this includes induction
of oral tolerance, potent anti inflammatory effects, mucosal IgA expression and
effects on epithelial cell proliferation and differentiation. TGF- is also reported to
have vital effects for the differentiation and development of FOXP3+ regulatory T
cells (Tregs) [28] which have a three fold role of immune tolerance by utilize TGF-
to halt cell activation and differentiation that leads to immune suppression; immune

stimulation by converting immature T cells into iTregs and Th17 to reinforce for their
action against inflammation and pathogenic microbes[11] ; and finally fighting
apoptosis and destabilization as Tregs do their job.[12] Because of this, it is
proposed that EPS stimulates or inhibits immune response via TGF-B. The
mechanism of this process is proposed by two models : Model 1 which proposed the
presence of a single EPS receptor with co stimulants for stimulation and inhibition
which guides the pathway that it will transcend and Model 2 which proposes
different receptors for immune inhibition and stimulation with an unknown sorter
that sorts signal and guides process to be activated.

Figure 1. Model 1: Presence of single EPS receptor with co stimulant for

immune stimulation and inhibition

It is not clear whether EPS is recognized via pattern recognition receptors or

have a unique receptor for it. Model 1 proposes a certain EPS receptor that
activates TGF beta and has effects directed by costimulants that either

inhibit or stimulate immune response. Stimulation of Costimulant S invoke

signal transduction of either NF-kb pathway and JNK pathway. This
hypothesis is based upon the model by Horbelt et al about the pathways
induced by TGF-Beta and Probiotic immune studies that suggest TGF beta
involvement in the process and also, the upregulation of various molecules in
the said pathways.

Figure 2. Model 2: Different receptors for immune inhibition and stimulation

with an unknown sorter

On the other hand, Model 2 suggests different receptors for immune

inhibition and stimulation. These in turn cascades different effectors that
activates TGF-beta and activates either pro or anti inflammatory responses
based on the receptor that is activated. There is no solid evidence that could
backup the hypothesis on the receptors. This could only be proved by gene

silencing studies that would demonstrate that the absence of a certain gene
that codes for a possible receptor would elicit negative effects on its
probable usual uses. Receptor and EPS binding should also be done in order
to prove interactions of the two.

EVALUATION OF THE HYPOTHESIS

Immune stimulation for Fighting pathogens
In order to fight potential infectious pathogens, Probiotics can induce innate
nonspecific cellular immune response through activation of natural killer (NK)
cells, macrophages, antigen-specific cytotoxic T-lymphocytes, and cytokine
release that are strain-specific and dose-dependent. In addition,the mixture
and type whether gram-positive or negative may also induce different
cytokine responses. [9] Studies show that BALB/c mouse pups fed with
Lactobacillus casei before infection with influenza virus toned down viral
titers in nasal lavage fluid and at the same time increased lung natural killer
cell activity, and consequently tripled survival rate.[25] The feeding of L
rhamnosus to BALB/c mice also inreased respiratory antiviral immunity
through Toll-like receptor 3 activation.[24] which are regulated by Th1 and
Th17 cytokines affected by TNF-beta. [26] However, a study using L. acidophilus

has done the opposite and significantly inhibitted NF-B activation stimulated
by S. typhimurium. IL-8 mRNA when CaCO2 cells were pretreated when
compared with those infected with S. typhimurium all by itself. In addition,
synbiotics showed stronger suppresion on IL-8 and TNF- expression
elevated by S. typhimurium. The suppression is based on increased IB
expression level and Smad3/4 activity, molecules of the TGF B pathway. [30]
Immune suppression for tolerance for harmless antigens
On the other hand hand, Probiotics could also inhibit local innate immune
system by clonal deletion or anergy, in high doses of antigen ingested or
active suppression after induction of regulatory T cells in Peyer's patches
[10]. In a study by Zhen B, in vitro, with human peripheral blood
mononuclear cells (PBMC), and in vivo, with murine dextran sodium sulfate
(DSS) colitis model They found out that B. breve incubation was successful in
inhibiting Th1 and promote Treg cell population unlike L. rhamnosus which conferred
no effects. [23]

Distinct Immune responsiveness to antigens

In addition to the type of antigen, some other factors also play a role in production
of distinct immune response to antigens and those are the path of antigen entry,
the dose,the age of the host and the timing of the introduction. Upon antigen
inreoduction, host immune cells release a host of cytokines that directs the further
immune responses. However, putting all things the same, except for type of

antigen, it was reported that T helper 1 and T helper 2like cells produce
highly polarized patterns of cytokines [21]
Ability of EPS to function as immune modulator by itself
A study by C. Hidalgo et al showed the capabilities of the EPS of various
Lactic acid bacteria and Bifidobacteria to elicit immune modulation. They
have found out that EPS having negative charge and/or small molecular
weight can have mild stimulatory effects on immune cells, while uncharged
polymers and have high molecular weight have suppressive capabilities.[2]
There is also a study that reported in hyporesponsiveness in macrophages,
after incubation with LAB EPS, decreasing levels of tumor necrosis factor
(TNF-), IL-12 and interleukin 6 (IL-6) upon reintroduction of the EPS and
Lipoteichoic acid. [3]

Induction of TGF-B by Probiotics

In a study by of 19 preterm infants which using Bifidobacterium breve
supplementation show increased Serum TGF-beta1 on day 14 which
remained until day 28. In addition, a TGF-beta1 feedback inhibitor Smad7
expression was reduced while a signal transductor Smad3 had enhanced
expression on day 28. [22] The NF-kB pathway induction is supported by a

study using L. acidophilus which shows significant inhibition of NF-B

activation stimulated by S. typhimurium.[30]
CONSEQUENCES OF THE HYPOTHESIS AND DISCUSSION
If it is found out that EPS of probiotics alone could stimulate the balance o f immune
stimulation for fighting against pathogens and immune tolerance for harmless
antigens, then EPS research for this area is needed to classify the effects of EPS
structure and composition and their corresponding immune effects. In addition this
could provide an option if ever to harness the health benefits of Probiotics without
introducing the organism itself, which makes way for more ways of delivery and a
promise of more efficacy as probiotics are constantly in danger of extermination by
antibiotics or when introduction of beneficial probiotics are impossible.
On the other hand, transforming growth factor-beta (TGF-) has a vital role in
epithelial cancers, in a study with head and neck squamous cell carcinoma,
inhibition of TGF- signaling increased de novo tumor growth, while TGF-
overexpression and transduction promotes malignancy. They have also observed
that this effect is associated with aberrant activation of nuclear factor-B (NF-B)
whose cross-talk is mediated through TAK1 and SMAD7. This proposes another
possibility of another pathway by which probiotics could inhibit and affect cancer
development and progression [27].

REFERENCES
[1] Nuria Salazar,1 Patricia Lpez,2 Pablo Garrido,3 Javier Moran,3 Estefana
Cabello,3 Miguel Gueimonde,1 Ana Surez,2 Celestino Gonzlez,3 Clara G. de
los Reyes-Gaviln,1 and Patricia Ruas-Madiedo1 Immune Modulating
Capability of Two Exopolysaccharide-Producing Bifidobacterium Strains in a
Wistar Rat Model http://dx.doi.org/10.1155/2014/106290

[2] Hidalgo C. Patricia Lopez Miguel Gueimonde Clara G. De los ReyesGaviln Ana Suarez Abelardo Margolles Immune Modulation Capability of
Exopolysaccharides Synthesised by Lactic Acid Bacteria and Bifidobacteria
Probiotics and Antimicrobial Proteins 12/2012; 4(4). DOI: 10.1007/s126020129110-2
[3] Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, et al.
Induction of colonic regulatory T cells by indigenous Clostridium species.
Science 2011;331:337-41.
[4]. Karimi K, Inman MD, Bienenstock J, Forsythe P. Lactobacillus reuteriinduced regulatory T cells protect against an allergic airway response in
mice. Am J Respir Crit Care Med 2009;179:186-93.
[5] MARTA CISZEK-LENDA1, BERNADETA NOWAK1,MALGORZATA
SROTTEK1,MARIAWALCZEWSKA1,SABINA GORSKA-FRACZEK2, ANDRZEJ
GAMIAN2, JANUSZ MARCINKIEWICZ1 Further studies on immunomodulatory
effects of exopolysaccharide isolated from Lactobacillus rhamnosus KL37C
DOI: 10.5114/ceji.2013.37743
[6] Mark F. Cesta. Normal Structure, Function, and Histology of MucosaAssociated Lymphoid Tissue. doi: 10.1080/01926230600865531 Toxicol
Pathol August 2006 vol. 34 no. 5 599-608
[7] Gormley PD, Powell-Richards AO, Azuara-Blanco A, Donoso LA, Dua HS
(1998) Lymphocyte subsets in conjunctival mucosa-associated-lymphoidtissue after exposure to retinal-S-antigen. Int Ophthalmol 22:7780.
[8]
Croitoru K, Bienenstock J(1994) in Handbook of Mucosal Immunology,
Characteristics and Functions of mucosa-associated lymphoid tissue, eds
Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee JR, Bienenstock J
(Academic Press, San Diego), pp 14151
[9] Ashraf R1, Shah NP. Immune system stimulation by probiotic
microorganisms. Crit Rev Food Sci Nutr. 2014;54(7):938-56. doi:
10.1080/10408398.2011.619671.
[10] Erika Isolauri, Yelda Stas, Pasi Kankaanp, Heikki Arvilommi, and
Seppo Salminen Probiotics: effects on immunity1,2,3 Am J Clin Nutr February
2001 vol. 73 no. 2 444s-450s

[11] Simone Kennedy Bedoya, Brandon Lam, Kenneth Lau, and Joseph Larkin III
Th17 Cells in Immunity and AutoimmunityClinical and Developmental Immunology
Volume 2013 (2013), Article ID 986789, 16 pages
http://dx.doi.org/10.1155/2013/986789
[12] Dat Q. Tran TGF-: the sword, the wand, and the shield of FOXP3+ regulatory T
cells J Mol Cell Biol (2011) doi: 10.1093/jmcb/mjr033

[13] Khailova L, Frank DN, Dominguez JA, Wischmeyer PE. Probiotic administration
reduces mortality and improves intestinal epithelial homeostasis in
experimental sepsis.Anesthesiology2013,119(1):166-177.
[14]Forsyth CB, Farhadi A, Jakate SM, Tang Y, Shaikh M, Keshavarzian A.
Lactobacillus GG treatment ameliorates alcohol-induced intestinal oxidative
stress, gut leakiness, and liver injury in a rat model of alcoholic
steatohepatitis.Alcohol2009,43(2):163-172
[15] Vinolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation
by Short Chain Fatty Acids. Nutrients2011,3:858-876
[16] Schlee M, Harder J, Koten B, Stange EF, Wehkamp J, Fellermann K. Probiotic
lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp
Immunol2008,151(3):528-535
[17] Thomas CM, Versalovic J: Probiotics-host communication. Modulation of
signaling pathways in the intestine.Gut Microbes2010,1(3):148-163.
[18]Bermudez-Brito M, Plaza-Diaz J, Munoz-Quezada S, Gomez-Llorente C, Gil A:
Probiotic Mechanisms of Action. Ann Nutr Metab2012,61:160-174
[19] Villena J, Kitazawa H. Modulation of intestinal TLR4-inflammatory signaling
pathways by probiotic microorganisms: lessons learned from Lactobacillus
jensenii TL2937. Front Immunol2014; 4(512):1-1
[20] Timmerman HM, Koning CJ, Mukder L, Rombouts FM, Beynen
AC.Monostrain, multistrain and multispecies probioticsAcomparison of
functionality and efficacy.Int J Food Microbiol2004,96(3):219-233.
[21] Mossman TR, Cherwinski H, Bond MW, Gielin MA, Coffman RL. Two types of
murine helper T cell clone. I. Definition according to profiles of lymphokine activities
and secreted proteins. J Immunol 1986;136:234857.
[22] Fujii T1, Ohtsuka Y, Lee T, Kudo T, Shoji H, Sato H, Nagata S, Shimizu T,
Yamashiro Y. Bifidobacterium breve enhances transforming growth factor beta1
signaling by regulating Smad7 expression in preterm infants. J Pediatr Gastroenterol
Nutr. 2006 Jul;43(1):83-8.
[23] Zheng B1, van Bergenhenegouwen J2, Overbeek S1, van de Kant HJ1, Garssen
J2, Folkerts G1, Vos P3, Morgan ME1, Kraneveld AD1. Bifidobacterium breve
attenuates murine dextran sodium sulfate-induced colitis and increases regulatory T
cell responses.PLoS One. 2014 May 2;9(5):e95441. doi:
10.1371/journal.pone.0095441. eCollection 2014.
[24] Kitazawa H, Villena J. Modulation of respiratory TLR3-anti-viral response by
probiotic microorganisms: lessons learned from Lactobacillus rhamnosus CRL1505.
Front Immunol 2014;5:201.
[25] Yasui H, Kiyoshima J, Hori T. Reduction of influenza virus titer and protection
against influenza virus infection in infant mice fed Lactobacillus casei Shirota. Clin
Diagn Lab Immunol 2004;11:675-9.

[26] Tournadre A1, Lenief V, Miossec P. Expression of Toll-like receptor 3 and Toll-like
receptor 7 in muscle is characteristic of inflammatory myopathy and is differentially
regulated by Th1 and Th17 cytokines.Arthritis Rheum. 2010 Jul;62(7):2144-51. doi:
10.1002/art.27465.
[27] Freudlsperger C1, Bian Y, Contag Wise S, Burnett J, Coupar J, Yang X, Chen Z,
Van Waes C. TGF- and NF-B signal pathway cross-talk is mediated through TAK1
and SMAD7 in a subset of head and neck cancers. Oncogene. 2013 Mar
21;32(12):1549-59. doi: 10.1038/onc.2012.171. Epub 2012 May 28.
[28] Horbelt D, Denkis A, Knaus P (2012) A portrait of Transforming Growth Factor
superfamily signalling: Background matters. Int. J. Biochem. Cell Biol. 44(3), 46974.
[29] Dardalhon V., Korn T., Kuchroo V.K., et al . Role of Th1 and Th17 cells in organspecific autoimmunity. J. Autoimmun. 2008b;31:252-256.
[30] Da-Yong Ren,1,2 Chang Li,1 Yan-Qing Qin,1,2 Rong-Lan Yin,3 Shou-Wen Du,1
Fei Ye,1 Hong-Feng Liu,1 Mao-Peng Wang,1 Yang Sun,1 Xiao Li,1 Ming-Yao Tian,1
and Ning-Yi Jin1 Lactobacilli Reduce Chemokine IL-8 Production in Response to TNF and Salmonella Challenge of Caco-2 Cells BioMed Research International Volume
2013 (2013), Article ID 925219, 9 pages http://dx.doi.org/10.1155/2013/925219