Volume 28 Number 2 (2013) Independent thoughts for independent minds

International

PigTopics

GUT HEALTHThe importance of intestinal cells.

NUTRITIONReviewing the nutrient needs of modern pigs.

PERFORMANCEPiglets benefit from monoglycerides in sow’s milk.

ENVIRONMENTAL CONTROLWe look at options from around the world.

SALMONELLAControl and monitoring tailored to specific needs.

PCV2 VACCINATIONLong lasting protection favours batch homogeneity.

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International Pig Topics — Volume 28 Number 2

by R. Davin, Grup de Recerca enNutrició, Universitat Autònomade Barcelona, 08193 Bellaterra,Spain.

There are basically two typesof cell cultures: (a) primarycell cultures and (b) continu-

ous cell lines. Primary cells aredirectly isolated from a subject thathave limited lifespan, whereas con-tinuous cell lines, also referred asestablished or immortalised, haveacquired the ability to proliferatewith infinite lifespan.

There are several means toachieve the immortality of a cellline: tumor cells and undifferenti-ated cells are inherently immortal,otherwise normal cells can beimmortalised (confer tumoral abil-ity) adding foreign genes through avirus.

Continuous cell lines form whatthe scientific community designateas cell lines and are one of the mostused and useful models to study theeffect of a specific factor (for exam-ple feed additives, drugs, micro-organisms) on a specific cell type(epithelial cells, lymphocytes,fibroblasts) at the most basic level.

Cells are seeded in a plate and aregrown and maintained at an appro-priate temperature, gas mixture andgrowth media.

IPEC-J2 is a non-transformedepithelial cell line isolated from aneonatal piglet that has been andstill is used as a model of experi-

mental challenges with pathogens,such as salmonella, E. coli and otheragents.

In this article we explain the use ofIPEC-J2 cell line as a study modelfor feed ingredients and additives inthose challenging (infectious) situa-tions.

What are IECs?

The surface of the intestinal epithe-lium represents an area of approxi-mately 100m2. It consists of a singlelayer of columnar epithelial intesti-nal cells (IECs) that separates theintestinal lumen from the externalenvironment of the host tissue.

IEC’s are involved in multiple func-tions of the digestive system, suchas the absorption of nutrients andwater, exchange of electrolytes andhormone production.

Because of its permanent exhibi-tion to the external environment,IECs are of key importance formaintaining the integrity of theintestinal epithelium and reducingthe risk of invasion by externalagents, forming a physical barrier.They are also crucial for the mainte-nance of intestinal homeostasis.

Epithelial intestinal cells help onthe physical barrier function due totwo physical structures of theepithelium (microvillus and tightjunctions) that separates the exter-nal environment of the organism,preventing the invasion of micro-

organisms. IECs also secrete a largevariety of antimicrobial peptides,cytokines and chemokines that areactivated in pathological or infec-tious situation enhancing the inflam-matory response.

During an infection, when apathogen breaches the intestinalmucosa it is recognised by toll-likereceptors (TLRs). TLR areexpressed at the intestinal surfaceplaying a key role in the innateimmune system, since they activateimmune cell responses.

Between the IEC there are globetcells that secrete mucus. This mucuscontains mucins that form a perme-able barrier that protects andrepairs the intestinal epithelium ofany damage. Mucins can also inter-act with bacteria and have the abilityto immobilise and eliminate themthrough the movements of the gut.

Intestinal cell lines

Most research has been conductedin human intestinal cell lines that arederived from a cancerous colon (forexample HT-29, T84, Caco-2) or

duodenum (HUTU-80). Also insmall intestinal rat cell lines (forexample IEC-6 and IEC-18), whichare not cancerous.

However, the structure and func-tion of the gastrointestinal tract ofrodents appear to differ from thatof other domestic mammals likeswine.

Nowadays, there are three differ-ent porcine intestinal epithelial celllines: l IPI-2I cells were isolated fromileal tissue of an adult boar andwere transformed with a plasmidthat immortalises the cells. l IPEC-1 cells were isolated from amixture of ileal and jejunal tissue ofa day old piglet. It is a non-trans-formed cell line. l IPEC-J2 cell line was isolatedfrom jejunal tissue of a day old pigletand is non-transformed.

The lack of intestinal epithelial celllines not derived from tumors andthe specificity for swine stands theimportance of IPEC-J2 cell line, fur-ther considering that it can appro-priately support infections by a largevariety of micro-organisms. Most

Continued on page 9

Importance of intestinalcells for gut health –IPEC studies

Fig. 1. IPEC-J2 cells attached at the well-plate surface.

Fig. 2. Barrier function of IECs (J. R. Turner, 2008).

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importantly, because it has beendemonstrated that results of studiesperformed with IPEC-J2 cells can betranslated to porcine whole animalstudies.

How IPEC are affected

IPEC-J2 has become more usedsince the characterisation done byShierack et al., 2006. Since then,more than 15 articles havedescribed the expression of differ-ent cellular and immune molecules,and more than 20 articles have usedthem as a model of the intestinalepithelium for micro-organisminfections.

Before its characterisation, IPEC-J2 cells were used as a model ofporcine proliferative enteropathy(Lawsonia intracellularis) infectionbecause it internalises and prolifer-ates on them, as it was alsoobserved in chlamydia challenges.

When Shierack et al., 2006 char-acterised IPEC-J2 cells, they demon-strated that the IPEC also supportinvasion of different species of sal-monella and adhesion of E. coli.

The ability of these bacteria toinduce an inflammatory and immuneresponse (for example expressionof cytokines and TLRs) has beenrepeatedly demonstrated in follow-ing studies with IPEC-J2. It has alsobeen shown that the permeability ofIPEC-J2 monolayers (measured bytrans-epithelial electrical resistance;TEER) is reduced by enterotoxi-genic Escherichia coli (ETEC).

Escherichia coli, salmonella andchlamydia are important pathogensin the swine industry as well as rep-resentatives with highly differentinfection strategies; that is one ofthe most important reasons whyIPEC-J2 cell line represents anexcellent model for the interactionsof these pathogens with the porcineintestinal epithelia.

ETEC toxins have also been com-pletely studied on IPEC-J2, theirroles in bacterial adherence havebeen presented by Johnson et al.,2009. Adherence is a key point asthe adhesion of ETEC to the intesti-

nal mucosa is a prerequisite step forits colonisation.

More recently, IPEC-J2 cells wereused to demonstrate that high con-centrations of deoxynivalenol, atoxin released by the fungus fusar-ium, reduced the viability of porcineintestinal epithelial cells. This isinteresting on swine nutritionbecause deoxynivalenol often cont-aminates cereal grains, which causediarrhoea and weight loss in pigs.

The IPEC-J2 cells have beenincreasingly used to study potentialprobiotic micro-organisms focusingon either the adhesiveness of theprobiotic to IPEC-J2 cells or theability of the probiotic to inhibit theinflammatory response induced by aspecific pathogen.

Some examples of these probi-otics are Enterococcus strains,Saccharomyces strains or multiplespecies of lactobacillus. The validityof these studies remains to be seenas articles that use the tested probi-otics in vivo have yet to be pub-lished.

In a recent publication differentfeedstuffs (wheat bran, casein glyco-macropeptide, mannan-oligosaccha-rides, locust bean extract andAspergillus oryzae fermentationextract) were able to reduce theadhesion of ETEC to IPEC-J2 andinterfere on the innate inflammatoryresponse. Another study demon-strates that phytic acid decreased

the negative effects of deoxyni-valenol on the membrane integrityof the intestinal epithelial cell line.Among the various feedstuffs oradditives used in IPEC studies, wewill merit special attention to thoserelated to the use and influence ofzinc in the intestinal epithelium.

Zinc dose and source

Use of in-feed pharmacological lev-els of zinc oxide (ZnO, 2500-3000ppm) is widely accepted in thepig industry to reduce the incidenceand severity of post-weaning diar-rhoea in piglets, in particular thatcaused by ETEC K88.

However, the therapeutic mecha-nism is not yet well understood. In afirst in vitro study conducted withthe human intestinal cell line Caco-2it was demonstrated that increasingconcentrations of ZnO reducedETEC K88 adhesion and internalisa-tion to the cell line. It was alsofound that ZnO prevent theincrease in tight junction permeabil-ity induced by ETEC and the ETEC-induced expression of severalinflammatory response genes.

This study did not show an antimi-crobial effect of ZnO, and providesalternative mechanisms for thereduced incidence of post-weaningdiarrhoea in ZnO supplementedpigs. However, as stated above,

Caco-2 is a human intestinal cellline, while ETEC K88 is a porcine-specific pathogen. Some recentstudies aimed to investigate theeffect of ZnO dose and source onIPEC-J2 challenged with ETEC,approaching the investigation to abetter research porcine model.IPEC-J2 cells were used as an invitro model of intestinal ETEC infec-tion with or without supplementedZnO.

Genomic analysis identifiedincreased expression of a variety ofinnate immune response genes (forexample cytokine genes) inresponse to ETEC exposure but itwas reduced when cells were simul-taneously exposed to ZnO.

Metabolic effects showed thatZnO may modify IPEC-J2 cell mor-phology at low concentrations, andreduces cell viability at higher val-ues. When ETEC adhesion to thehost cell was measured after onlytwo hours, ZnO supplementationdid not show any effect.

However, when it was measuredon a longer period, ZnO at concen-trations which do not affect IPEC-J2morphology decreased ETEC adhe-sion. These findings suggest that thedose and source of ZnO have a sig-nificant impact on both IPEC-J2 andpathogen metabolism and alsoreduce the expression of inflamma-tory genes when IPEC-2 were chal-lenged.

In conclusion:l Intestinal epithelial cells are thefirst line of defence from the exter-nal environment and also have animportant role on the inflammatoryresponse. l The use of epithelial cell lines asan in vitro model has been increas-ing in recent years. l IPEC-J2 cell line provides a goodspecific swine model to study theeffect of feedstuffs on the inflamma-tory response caused by specificporcine pathogens. l The use of IPEC-J2 and other celllines is expected to increase in thefuture. IPEC-J2 can be an excellentmodel to investigate the ZnO thera-peutic mechanism. n

References are available from the author on request

Continued from page 7

Fig. 4. Salmonella can invade epithelial cells (step 1). This intracellularinfection leads to a local inflammatory response (step 2) mediated byneutrophils (step 3), with subsequent tissue damage and epithelialsloughing (step 4). (Gonzalez-Escobedo et al., 2011).

Fig. 3. Summary of the cytokines and chemokines produced by IECs.

100

98

96

94

92

90

%mg/l ZnO

0.75 1.5

Regular ZnO

Potentiated ZnOHiZox* Animine

Fig. 5. Reduction of ETEC adhe-sion to IPEC-J2 cells.

Reprinted from International Pig Topics, published by Positive Action Publications Ltd, PO Box 4, Driffield, East Yorkshire YO25 9DJ, UK. Tel: +44 1377 241724 Fax: +44 1377 253640 Email: info@positiveaction.co.uk

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