September | October 2013

Marine algal polysaccharides: a new option for immune stimulation

The International magazine for the aquaculture feed industry

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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Nowadays, all animal productionis concerned with vaccination.This is an essential techniquefor the protectionof livestock

health which, however, entails significantcosts for stock breeders. Maximizing theefficiency and profitability of prophylacticvaccination strategies is therefore a majorstake. To achieve this, new avenues areconstantlyexplored.Oneoftheseconcernsistheuseofnewmoleculesextractedfromseaweeds to help optimize the stimulationof thenaturaldefencesof thebodyand itsresponsetovaccinationstrategies.

Innate immunityThebody’sresponsetotheaggressionofa

pathogenisbasedontwotypesofimmunity.Theyaretheinnateimmuneresponseandtheadaptiveresponse.

The innate response is the first line of

defence against pathogens. It is activatedimmediately and acts very quickly. Thisimmune response can be found in all ani-mals. It will be the same whenever thebody encounters that pathogen. However,the body does not retain a memory of theinfectiousagent.Themechanismofactionofthis type of immunity consists in recognizingthe molecular patterns shared by numerouspathogens,which are essentially representedbymembranefractions(glycocalyx).

The various elements that contribute totheinnateimmuneresponsearethefollowing:

• Physical barrier (mucous membrane,skin,mucus,villietc)

• Phagocyticcells,suchasthemacrophages• Naturalkiller(NK)cells• Certain cytokines, which deliver signals

warningthebodyofadanger• Complementsystem• Toll-like receptors (TLR), a family of

membrane receptors only discoveredrecently. They control the expressionofmoleculesthatfightagainstinfectiousagents(directlyorindirectly,viaeffectorcells,andbyrecruiting theactivationoftheadaptiveimmunesystem).

The elements associated with the innateimmune response can act on the pathogendirectly or indirectly, by producing effectorcells (cytokines etc). The latter will subse-quently trigger the adaptive immunity byactivatingtheTandBcells.

Adaptive immunityUnliketheinnateresponse,theacquiredor

adaptiveresponseoccursinvertebratesonly.Duringthefirstencounterwithagivenpatho-gen (primary infection), it acts as the body’ssecond line of defence. Its activation takessometime-knownaslatency.However,thisresponse system memorizes the pathogensit encounters and when the body is againexposedtothemthelatencyismuchshorterandtheimmunesystemreactstotheaggres-sion almost immediately. Adaptive immunityisspecific:itrecognisesthemolecularpatternsofthealreadyencounteredpathogens.

The various elements that contribute tothe adaptive immune response are the fol-lowing:• Tcells• Bcells• Antibodies• Ig, TCR,CTL, antibody (AB)-producing

plasmacells+coupledaidoftheinnateimmunityeffectors

Seaweeds: a new source of active elements to stimulate the immune system

In recent years more and more publica-tions have brought to the forefront therelevanceofseaweedsinnumerousbiologicalapplications, particularly to immune mecha-nisms,takingspecial interest insomeoftheircomponents, namely the sulfated polysac-

Marine algal polysaccharides: a new option for immune stimulation

18 | InternatIOnal AquAFeed | September-October 2013

FEATURE

September-October 2013 | InternatIOnal AquAFeed | 19

NK : Natural KillerPRR : Pattern Recognition Receptor

CMH : Complexe Majeur d’histocompatibilitéTCP : T Cell Receptor

64 | InternatIOnal AquAFeed | September-October 2013

charides. These are complex carbohydrateswhichdonotoccurinterrestrialplants.Theyaresupposedtoinfluencetheimmunesystemby a vast number of still poorly understoodpathways.

Polysaccharides represent a structurallydiverse class of macromolecules which arerelatively widespread in nature. There aresimpleandcomplexformsofpolysachharides.Unlike proteins and nucleic acids, polysac-charidescontain repetitivestructural featureswhicharechainsofmonosaccharideresiduesjoinedtogetherbyglycosidicbonds.

Thus,theyformpolymer(-type)structuresrepresentedintheformofchainsthatmaybe

homogenous (homopolysaccharides) or not(heteropolysaccharides). The simple formsare the homopolysac-charides composed ofa single type of sugar,linked in an essentiallylinear manner (starch,glycogen, cellulose forexample). They areessentially structuralcompounds or mecha-nismsofenergystoragein an easily releasableform. Their structuremay become more

complex owing to their capacity toestablish links at various levels of eachelementary unit, allowing thus thedevelopment of branching structuresinthethreedimensions.Thesearethebranchedheteropolysaccharides.

Structural variability and biological potentialities

Thenucleotidesinnucleicacidsandthe amino acids in proteins can inter-connect in only one way, while themonosaccharide units in oligosaccha-ridesandpolysaccharidescanintercon-nect at several points to form a widevarietyof linearorbranchedstructures(Sharon and Lis 1993). For instance,

the number of possible permutations forfour different sugar monomers can attain

18 | InternatIOnal AquAFeed | September-October 2013 September-October 2013 | InternatIOnal AquAFeed | 19

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Innate immunity / acquired immunity: two complementary and cooperative systems

Innate immunity adaptive or acquired immunity

ChronologyPrimary infection Quick response: first

barrier against pathogens Second line of defence : latency

(about 7 days)

repeated infections Identical to the primary response

Immune memory => latency close to zero

Specificity non-specific response Specific response (Ig and tCr)

recognized molecular patterns Invariable and common to numerous pathogens Specific to the infectious agent

Cellular and molecular effectors Complement, phagocytic cells and certain cytokines

Ctl (cytotoxic t cells) and anti-body producing plasma cells,

with the help of innate effectors

tCr : t Cell receptor - Ig : Immuno Globulin - Ctl : Cytotoxic t lymphocyte or Killer t cells

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up to 35,560 unique tetrasaccharides, whilefour amino acids can form only 24 differentpermutations(Hodgson1991).

Thisexplainsthe factthat,amongmacro-molecules, polysaccharides provide the high-est capacity for carrying biological informa-tion, as they have the greatest potential forstructural variability. In addition, one of theparticularities thatnumerousmarinepolysac-charidespossessistheirpolyanioniccharacter,whichconfersthemahighchemicalreactivity.

Of these anionic polysaccharides, themajority of those which occur macroalgaeare sulfated polysaccharides: galactan (agar,carraghenans),ulvans,fucans.

Theulvans,forexample,thewater-solublepolysaccharides found in green seaweed oftheorderUlvales (Ulva and Enteromorpha),have sulfate, rhamnose, xylose and iduronicand glucuronic acids as their main constitu-ents (Lahaye and Ray 1996) (Percival andMcDowell1967).

Ulvan structure shows great complexityandvariabilityasevidencedbythenumerousoligosaccharide repeating structural patternsidentified(LahayeandRobic2007).Themainrepeating disaccharide units reported are ofulvanobiouronicacid3-sulfatetype,containingeitherglucuronicoriduronicacid.Inaddition,a few repeating patterns can be found that

contain sulfated xylose replacing uronic acidorglucuronicacidontheO-2binding/linkoftherhamnose-3-sulfateunits(LahayeandRay1996)(Lahayeetal.1997).

InterestsThis huge variability in thepolysaccharide

structure provides the flexibility required forexactregulatorymechanismsindifferentcell-cellinteractionsinhigherorganisms.

Sulfation in particular seems to be con-ducive to various biological activities notedin polysaccharides extracted from marinemacroalgae.

Marine sulfated polysaccharides: their role and effect on immunity

Sulfated polysaccharides, which are wide-spread in macroalgae, have been shownto possess anti-infectious (Cumashi et al.2007)(WitvrouwandDeClercq1997)(anti-viral, anti-bacterial, anti-tumoral), antioxidant(Wangetal.2010)(deSouzaetal.2007)andanti-thrombotic (Mao et al. 2006) activities,as well as immune-modulating (Leiro et al.2007) activities that might find relevance instimulating the immune response or in con-trollingtheactivityofimmunecellsinordertomitigatenegativeeffectssuchasinflammation(Chen et al. 2008) One of the pathways ofmarine sulfated polysaccharides, which hasbeenemphasizedrecently,istheirroleintheactivationofTLR.

Indeed,moreandmorestudiesaredemon-stratingthatmarinealgalpolysaccharidescaninflu-encetheinnateimmuneresponsebybindingtorecognitionreceptorscalledPatternRecognitionReceptorsc (PRR), suchas themannose recep-tors or TLRs of phagocytic cells, including andespeciallymacrophages(Chenet al.2008).TLRsaretransmembraneproteinswhichdetectinvad-ingpathogensbybindingtoancestralmoleculesof microbial origin called Pathogen-AssociatedMolecularPatterns(PAMPs).

ThePAMPscontactatTRLleveltriggersacascadeofresponsesresulting intheexpres-sionofinflammatoryresponsegenes.Inmam-mals,theserecentlyidentifiedreceptorshavebeennumberedfrom1to11(TLR1-TLR11).

Oncontactwiththeirrespective PAMPs,TLR specifically acti-vateasignalingpath-way leading to theactivation of NF-kB(Nuclear Factor-kappa B) and AP1(ActivatorProtein1) transcription fac-tors regulating theexpressionof inflam-matory cytokinessuch as TNFα, IL-1orIL-6.

It therefore now

20 | InternatIOnal AquAFeed | September-October 2013

FEATURE

September-October 2013 | InternatIOnal AquAFeed | 21

Structure of the 4 main repeating ulvan patterns of Ulva lactuca

Ulvanobiouronic acid A. [→ 4)-β-D-GlcA-(1 → 4)-α-L-Rha3S-(1 →] n;

Ulvanobiouronic acid B. [→ 4) - α-L-IdoA-(1 → 4)-α-L-Rha3S-(1 →] n

Ulvanobiose A. [→ 4)-β-D- Xyl -(1 → 4)-α-L-Rha3S-(1 →] n;

Ulvanobiose B. [→ 4)-β-D- Xyl 2S-(1 → 4)-α-L-Rha3S-(1 →] n;

Classification of Marine Sulfated Polysaccharides (MSP)

Branched sulfated heteropolysaccharide

appears that TLR play a key role in the adaptive immune response, but the signals produced by their activation lead to the activation of numerous other cells and func-tions of the immune system, which makes them essential elements of both the innate immune mechanisms and of adaptive immu-nity.

The activity of some sulfated algal polysac-charides as TLR activating agents might be the result of a certain structural similarity between these marine polysaccharides and bacterial lipopolysaccharides (LPS). Bacterial LPSs are indeed a type of structure occurring at the surface of their external membrane and recognized as bacteria-specific recognition elements. In particular, bacterial LPS in mam-mals are shown to be specifically recognised by TLR4.

Possible applications in animal health

In conclusion, seaweeds appear to contain sugars in the form of polysaccharides, some of which - sulfated polysaccharides - are complex polyanionic structures which possess various biological properties. A vast number of studies have already evidenced the effects of some of these sulfated polysaccharides, particularly the fucoidans, the carraghenans and the ulvans, on certain mechanisms of inflammatory response and on immunity.

The identification and selection of these polysaccharides extracted from suitable macroalgae makes it possible to envisage the use of these molecules as agents for the stimulation of the various mechanisms associated with the body defence and, in particular, of the innate immunity mecha-nisms.

Within the framework of the potential applications in the fields of animal breeding and animal health two non-exclusive strategies can be proposed:

Regular sequential intakes for a general stimulation of the body’s state of defence: • With a regular intake not connected

with vaccination, they allow the

strengthen-ing of the body’s state of defence. Repeated use allows the development of a ‘basic’ immune sys-tem and the boosting of the state of defence of the innate system. The use of polysaccha-rides upstream

or downstream of a prophylactic programme may be an asset in enhancing the level of immune protection of an individual or group of indi-viduals within a livestock and in contributing to a better control of the infectious pressure on the livestock, preventing the appearance of recurrent infec-tious pathologies.

Targeted intakes within the framework of a vaccination pro-gramme:• As part of a

vaccination pro-gramme, they would enhance the vaccine protection. This would definitely provide the pos-sibility to improve the intake and persistence of the vaccine and there-by to improve the technical and economic per-formance of vac-cine prophylactic programmes.

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FEATURE

Sulfated polysaccharide

Cytokine/Chemokine production

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