Immunostimulatory activity of potential probiotic yeast strains in the dorsal air pouch system and the gut mucosa

ORIGINAL ARTICLE

Immunostimulatory activity of potential probiotic yeaststrains in the dorsal air pouch system and the gut mucosaA. Kourelis1, C. Kotzamanidis1, E. Litopoulou-Tzanetaki2, J. Papaconstantinou3, N. Tzanetakis3

and M. Yiangou1

1 Department of Genetics, Development & Molecular Biology, Biology School, Aristotle University of Thessaloniki, Thessaloniki, Greece

2 Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece

3 Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

Introduction

Immunity in the gut ecosystem is achieved by the interac-

tion of cells that participate in the architecture of the

gastrointestinal system with luminal microflora. Microor-

ganisms from the environment and the normal commen-

sal flora of the gastrointestinal tract have a marked impact

on the maturation of the mucosal immune system (Hessle

et al. 1999). They have been shown to crucially influence

the immune response establishment, especially the Th1

proinflammatory and Th2 ⁄ Treg anti-inflammatory and

regulatory balance (Wittig and Zeitz 2003). These

responses are mediated in part by the recognition of

microbe-associated molecular patterns (MAMPs) from a

class of pattern recognition receptors (PRRs), the Toll-like

receptors (TLRs), present on intestinal epithelial (IECs),

dendritic and macrophage cells (Neish 2002).

Probiotics are living microorganisms which upon

ingestion in adequate amounts confer health benefits to

the host (FAO ⁄ WHO 2002). Probiotics fulfil this defini-

tion through a variety of disparate and overlapping mech-

anisms such as regulation of the intestinal homeostasis,

antimicrobial activity and modulation of the innate

and ⁄ or adaptive immunity. The positive influence of

probiotics on the phagocytic activity of monocytes and

polymorphonuclear (PMN) cells as well as their effects on

IgA, chemokine and cytokine production after in vitro

and in vivo studies has been recently reviewed (Delcenser-

ie et al. 2008). These strain-specific beneficial probiotic

effects enhance the defence mechanisms and help the host

to maintain immune homeostasis (Galdeano et al. 2007).

The most extensively studied probiotics belong to the

genera Lactobacillus and Bifidiobacterium (Borriello et al.

2003; Tuohy et al. 2003). Despite the occurrence of yeasts

Keywords

air pouch, cytokines, gut mucosa, probiotic,

yeast.

Correspondence

Yiangou Minas, Department of Genetics,

Development & Molecular Biology, Biology

School, Aristotle University of Thessaloniki, 54

124, Thessaloniki, Greece.

E-mail: [email protected]

2009 ⁄ 1422: received 11 August 2009, revised

9 November 2009 and accepted 6 December

2009

doi:10.1111/j.1365-2672.2009.04651.x

Abstract

Aims: To determine the immunostimulatory activity of 15 presumptive

probiotic yeast strains in the dorsal air pouch system in comparison with their

activity in the gut mucosa.

Methods and Results: Presumptive probiotic yeast strains previously isolated

from human gastrointestinal tract and Feta cheese were further characterized

genotypically and biochemically. The Saccharomyces cerevisiae 982, Saccharo-

myces boulardii KK1 and Kluyveromyces lactis 630 strains exhibited in the air

pouch increased polymorphonuclear cell influx and phagocytic activity as well

as cytokine production with similar potency as the probiotics Ultra levure

S. boulardii and Lactobacillus acidophilus NCFB 1748. Oral administration of

these strains in mice results in differential activation of small intestine immune

responses concerning IgA and cytokine production as well as Toll-like receptor

expression.

Conclusion: Besides the Saccharomyces strains 982 and KK1, the K. lactis 630

strain could also be considered as a candidate probiotic.

Significance and Impact of the Study: The air pouch model may be used as an

alternative and rapid method for the discrimination and selection of potential

probiotic yeast strains.

Journal of Applied Microbiology ISSN 1364-5072

260 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271

ª 2010 The Authors

in many dairy-related products (Fleet 1990; Jakobsen and

Narvhus 1996) and the human gastrointestinal tract

(Psomas et al. 2001; Czerucka et al. 2007), their potential

as probiotics has been overlooked. So far, only yeasts of

the genus Saccharomyces are used as probiotics. Saccharo-

myces boulardii has been shown to possess immunomodu-

latory activity (Rodrigues et al. 2000; Czerucka et al. 2007)

and along with brewer’s yeast (Saccharomyces cerevisiae,

Saccharomyces carlsbergensis; Sargent and Wickens 2004)

are the only yeast strains commercialized as medicinal

probiotics in human medicine.

In previous studies, several yeasts were isolated from Feta

cheese and infants’ gastrointestinal tract (Tzanetakis et al.

1996; Andrighetto et al. 2000; Psomas et al. 2001), and

some of them were shown to possess in vitro probiotic

properties such as acid and bile tolerance and cholesterol

removal ability (Psomas et al. 2001, 2003). In vitro pro-

biotic assays could screen among a very high number of

microorganisms but cannot predict their behaviour in a

multifactorial in vivo system. In previous study, we have

demonstrated that the architecture of the epithelium

enclosed dorsal mouse or rat air pouch (Sedgwick et al.

1983) resembles gut mucosa concerning the immune

reactivity induced by potential probiotic Lactobacillus

strains (Kourelis et al. 2010a). Mice received Lactobacillus

strains in the air pouch or orally exhibited interaction

with the air pouch barrier and the gut mucosa barrier

and immune responses such as increased PMN chemo-

taxis and phagocytic activity as well as increased cytokine

production. We suggested that the air pouch system may

be used as an alternative and rapid in vivo method for

the initial discrimination and selection of potential pro-

biotic Lactobacillus strains (Kourelis et al. 2010a). These

results provide us with the biological system to examine

the in vivo immunomodulatory activity of potential

probiotic yeast strains.

On the basis of polyphasic taxonomy, it was of interest

to further characterize the above yeast strains based on

genotypic and biochemical methods and to determine

their potential immunomodulatory activity using the air

pouch model in comparison with the gut mucosa. We

present data that three yeast strains exhibiting immuno-

stimulatory activity in the air pouch also show regulation

of small intestine immune responses concerning the

production of IgA, cytokines and TLRs.

Materials and methods

Yeast and bacterial strains

The yeast strains (Table 1) were obtained from the collec-

tion of the Laboratory of Food Microbiology and Hygiene,

Aristotle University of Thessaloniki and were isolated from

Feta cheese and infants’ gastrointestinal tract (Tzanetakis

et al. 1996; Andrighetto et al. 2000; Psomas et al. 2001).

The strains 746, 832, 982, KK1, 570, 630, 414, KK2.1,

KK2.5, KK3.1, KK4.1, KK6.5, KK6P, KK5Y3 may be consid-

ered as potential probiotics because of their ability to with-

stand low pH values (pH 3Æ0) and high bile concentration,

reduce cholesterol levels and show adhesion capacity

(Psomas et al. 2001, 2003; Kourelis et al. 2010b). The

probiotic strains Ultra levure S. boulardii (UL; Biocodex,

Table 1 Biolog metabolic pattern identifica-

tion and pulsed-field gel electrophoresis

(PFGE) chromosome profile grouping of yeast

strains

Origin Strain Previous identification* Biolog

PFGE

grouping

Feta cheese 746 Saccharomyces cerevisiae Zygosaccharomyces cidri S. cerevisiae

832 No ID

840a Saccharomyces boulardii

952 S. cerevisiae

982 S. cerevisiae

Infants’ faeces KK1 S. boulardii S. boulardii S. boulardii

Ultra Levure UL

Feta cheese 570 Kluyveromyces lactis K. lactis K. lactis

630 Kluyveromyces marxianus

Feta cheese 414 Debaryomyces hansenii D. hansenii D. hansenii

Infants’ faeces KK2.1 Candida albicans C. albicans C. albicans

KK2.5

KK3.1

KK4.1

KK6.5 Candida parapsilosis C. parapsilosis C. parapsilosis

KK6P

KK5Y3 Isaatchenkia orientalis I. orientalis I. orientalis

No ID, no identification.

*(Andrighetto et al. 2000; Psomas et al. 2001; Tzanetakis et al. 1996).

A. Kourelis et al. Characterization of candidate probiotic yeasts

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271 261

Gentilly, France) and Lactobacillus acidophilus NCFB 1748

(National Collection of Food Bacteria, NCFB, Scotland)

were also included as positive control strains. The strain

S. cerevisiae 952 and the reference strains S. cerevisiae

NCYC167, Debaryomyces hansenii NCYC9 (National

Collection of Yeast Cultures: NCYC, Norwich, UK) and

Candida parapsilosis CBS1954 (Centraal Bureau voor

Schimmelcultures: CBS, Baarn, the Netherlands) were used

as negative controls because they exhibited no proven in vitro

or in vivo probiotic properties (Kourelis et al. 2010b).

Culture conditions and preparation of strains

Yeasts were grown at 30�C for 20–24 h in yeast extract

glucose peptone (YEGP) broth [2% (w ⁄ v) glucose, 0Æ5%

(w ⁄ v) yeast extract, 1% (w ⁄ v) peptone]. A YEGP agar

slope [2% (w ⁄ v) glucose, 0Æ5% (w ⁄ v) yeast extract, 1%

(w ⁄ v) peptone, 1Æ5% (w ⁄ v) agar] was used for the main-

tenance of the cultures at 4�C. L. acidophilus NCFB 1748

was maintained at )80�C in MRS (de Man, Rogosa,

Sharpe) broth with 25% (v ⁄ v) glycerol and was grown in

MRS broth at 37�C for 18 h. All strains were subcultured

at least three times prior to experimental use.

Yeast or NCFB 1748 cells were harvested from 20- to

24- and 18-h cultures, respectively by centrifugation and

washed twice with sterile saline. For administration into

the air pouch, yeast or NCFB 1748 bacterial cells were

resuspended in pyrogenic-free sterile saline at 5 · 107 and

5 · 108 CFU ml)1, respectively. For oral administration

in mice, yeasts were resuspended at 109 CFU ml)1.

Biolog YT microplate identification

Yeast strains were grown on Biolog Universal Yeast Agar

– BUY (Biolog Inc., Hayward, CA, USA) at 26�C for

48 h. Yeast identification was performed according to the

manufacturer’s instructions using the Biolog Microlog 3

computer software.

Pulsed-field gel electrophoresis (PFGE)

Chromosome plugs were prepared from 24-h yeast cul-

tures (109 cells per ml) according to the protocol of DNA

extraction as described previously (Steinkamp-Zucht and

Fahrig 1995) with an adaptation regarding the lyticase

enzyme concentration which was reduced in half for the

two Kluyveromyces strains.

PFGE karyotypes were performed in a Rotaphor R23

Tank (Biometra, Goettingen, Germany). Two different run-

ning conditions were used: duration 45 h, interval 50–20 s

linear, angle 130–115� log, field strength (124–114 V) log,

temperature 14�C in 1% (w ⁄ v) agarose for the S. cerevisiae

strains and duration 84 h, interval 350–50 s log, angle 110–

100� log, field strength 120–50 V linear, temperature 11�C,

in 0Æ7% (w ⁄ v) agarose for the other yeast strains. Agarose

gels were then stained with ethidium bromide while the

Saccharomyces cerevisiae YPH80 (New England Biolabs, Inc.,

Ipswich, MA) and Hansenula wingei (Bio-Rad Laboratories

Ltd, Herts, UK) strains served as molecular size standards.

The software GelCompar 4.0 (Applied Maths, Kortrijk,

Belgium) was used for normalization and processing of

the chromosome profiles and cluster analysis, based on

the Pearson coefficient and the unweighted pair group

algorithm with arithmetic averages (UPGMA).

Animals

Balb ⁄ c mice (25–30 g) and Fischer 344 rats (130–180 g)

aged 8–14 weeks were housed in stainless-steel cages with a

12-h light ⁄ dark cycle in a controlled atmosphere. The ani-

mals were fed ad libitum with a conventional balanced diet

and had free access to water during the experimental proto-

col. Animals were sacrificed by light ether anaesthesia fol-

lowed by cervical dislocation. All experiments were

performed in an accredited animal facility (number EL 54

BIO 02, School of Biology, Aristotle University of Thessalo-

niki). The protocol complied with the current ethical regu-

lations on animal research of our university and was in

accordance with both Greek National Legislation and to EC

Ethical Regulations. All groups included in the present

study consisted of five animals (unless otherwise indicated),

and each experiment was repeated at least two times.

Air pouch formation

Air pouches were raised on the dorsum of mice and rats

as we previously described (Kourelis et al. 2010a) by sub-

cutaneous injection of 3 and 20 ml sterile air, respectively.

Same size of air pouches was achieved by refilling the air

pouches with the appropriate volume of air day by day.

Six-day air pouches were then injected with 200 ll (mice)

and 1 ml (rats) of pyrogenic-free saline containing

5 · 107 CFU ml)1 of yeast or 5 · 108 CFU ml)1 of the

NCFB 1748 strain. Control animals received in the air

pouch only pyrogenic-free saline. The above doses were

chosen according to previous studies (Coates and McColl

2001; Kourelis et al. 2010a), and the fact that a beneficial

probiotic dosage is considered to be 107–109 CFU ml)1

(van Niel et al. 2002).

Determination of PMN accumulation in yeast or NCFB

1748-treated air pouches

The PMN cells accumulated in mouse or rat air pouches

in response to yeast or NCFB 1748 strain treatment were

harvested at the maximum of the induction 3 h post

Characterization of candidate probiotic yeasts A. Kourelis et al.

262 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271

ª 2010 The Authors

treatment (Kourelis et al. 2010a). PMN cells were col-

lected by centrifugation at 300 g for 10 min after injec-

tion in the rat or mouse air pouch of 10 or 2 ml of saline

respectively, followed by air pouch lavage. After centrifu-

gation, the supernatant was collected, filtered through

0Æ22-lm filter and hence referred to as the ‘air pouch

exudate’. The cell pellet containing the air pouch exudate

PMN cells was washed twice in saline and counted using

a haemocytometer while cell viability was determined by

trypan blue exclusion.

Phagocytic activity

Phagocytic experiments were performed using rat air

pouch PMN cells following the protocol of Loose et al.

(1978) with some modifications (Kourelis et al. 2010a).

Briefly, 100 ll containing 107 CFU ml)1 of baker’s yeast

(inactivated after boiling for 1 h) were co-incubated

(opsonized) with 100 ll of rat serum at 37�C for 30 min.

Then, 0Æ5 ml containing 5 · 106 cells per ml of air pouch

PMNs and 0Æ3 ml Hanks’ balanced salt solution (HBSS)

were added and incubated for additional 30 min. Samples

(100 ll) were taken at the indicated time post intervals of

5, 15 and 30 min. After stopping further phagocytosis by

adding 50 ll foetal bovine serum (FBS, Thermo Hyclone

Scientific, MA, USA), 50 ll trypan blue was also added to

visualize nonphagocytosed baker’s yeast cells. The number

of air pouch PMN cells containing baker’s yeast cells was

counted using a haemocytometer.

Detection of cytokines in air pouch exudates

Air pouch exudates were isolated from control or yeast-

treated air pouches and used for the determination of

tumour necrosis factor alpha (TNF-a), gamma interferon

(IFN-c) and interleukin 10 (IL-10). Determination of

cytokine concentration in mouse air pouch exudates was

performed using ELISA kits (eBioscience, Inc., San Diego,

CA, USA) according to the manufacturer’s instructions.

Purified cytokines included in the kit as well as air pouch

exudates isolated from bacterial lipopolysaccharide (LPS)

or Freund’s complete adjuvant (FCA) treated air pouches

were included as positive samples for the assay.

Histological samples

The yeast suspension (108 CFU per mouse) was orally

administered for 10 consecutive days to mice housed in

separate cages. The doses for oral administration used in

this study were chosen on the basis of the usual con-

sumption levels of 107–109 cells per ml for a probiotic to

have a beneficial effect (van Niel et al. 2002). The control

group received only pyrogenic-free sterile saline for the

same period. Small intestines were then removed, fixed in

4% parafolmadehyde in phosphate buffered saline (PBS)

and then embedded in paraffin blocks (Sainte-Marie

1962). Sections of 4 lm thickness were either stained with

haematoxylin–eosin or further processed for immuno-

histochemistry.

Immunohistochemical detection of IgA, cytokine and

TLR producing cells in the small intestine

The number of IgA and TLR2 producing cells was deter-

mined on histological sections by a direct immunofluo-

rescence assay. After deparaffinization and rehydration in

a graded series of ethanol, paraffin sections were incu-

bated with anti-IgA monospecific (Sigma, St Louis, MO,

USA) or TLR2 (Santa Cruz Biotechnology, Santa Cruz,

CA, USA) antibodies conjugated with fluorescein isothio-

cyanate (FITC) for 45 min and 2 h, respectively.

Cytokine, cyclooxygenase (COX) and TLR producing

cells were determined by an indirect immunofluorescence

assay. After deparaffinization and rehydration in a graded

ethanol series, paraffin sections were blocked with a 5%

(v ⁄ v) FBS in PBS solution for 30 min at room tempera-

ture (RT). The sections were then washed three times in

PBS and incubated for 2 h at RT with specific antibodies

directed against mouse TLR4, TLR9 (Abcam plc, Cam-

bridge, UK), TLR6, COX-1, COX-2, IL-5, IL-6, IL-12B

(Santa Cruz Biotechnology), IL-10, IFN-c and TNF-a(R&D systems Inc., Minneapolis, MN, USA). After four

washes in PBS, the sections were incubated for 1 h with

anti-goat or anti-rabbit (for IL-5) FITC-conjugated anti-

bodies (Sigma). The number of positive cells was counted

in 10 fields at 400· magnification. To confirm the speci-

ficity of the immunodetection, the samples were incu-

bated only with the secondary antibody, and the number

of positive cells was excluded from the number of cells

estimated for testing samples. For the final scoring, two

investigators achieved consensus.

Statistical analysis

Results are expressed as mean ± SEM. Multiple compari-

sons were performed by one-way anova followed by

Tukey’s test, and statistical significance was accepted at

values of P < 0Æ05.

Results

Biolog YT microplate identification and chromosome

length polymorphism (CLP)

Many of the probiotic properties are strain specific, and

thus reliable identification at strain level is required.

A. Kourelis et al. Characterization of candidate probiotic yeasts

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271 263

The yeast strains isolated from Feta cheese and infants’

gastrointestinal tract were identified by biochemical

(Application Programming Interface: API-ID32) and

molecular (randomly amplified polymorphic DNA PCR

and mitochondrial DNA restriction analysis) methods

(Tzanetakis et al. 1996; Andrighetto et al. 2000; Psomas

et al. 2001). On the basis of polyphasic taxonomy, these

strains were further characterized by Biolog YT microplate

identification and PFGE analysis. The data presented in

Table 1 show that Biolog YT method identified the strain

630, previously characterized as Kluyveromyces marxianus,

as Kluyveromyces lactis, while the strains 746 and 840apreviously characterized as S. cerevisiae were identified as

Zygosaccharomyces cidri and S. boulardii, respectively. The

strain 832 could not be identified (Table 1).

PFGE is considered as the gold standard method to

discriminate probiotics at strain level (FAO ⁄ WHO 2002).

PFGE analysis revealed polymorphism in the size and

number of chromosomes of the examined strains (Fig. 1).

The 746, 832, 840a, 952, 982 and KK1 strains had

chromosome band distribution (Fig. 1a) typical of yeast

species within the Saccharomyces sensu stricto complex

(Jespersen et al. 2000). The non-Saccharomyces strains

(Fig. 1b) showed a divergent chromosome profile accord-

ing to their species apart from the D. hansenii strains

(NCYC 9 and 414), which were clustered separately.

PFGE analysis of Isaatchenkia orientalis (KK5Y3) strain

(Fig. 1b) revealed only three bands ranging in size from

1Æ1 to 3Æ0 Mbp. The estimated chromosome number is at

least four, because of the increased fluorescence of the

�3Æ0 Mbp band. As far as we are concerned, this is the

first report of PFGE concerning I. orientalis.

Determination of the immunostimulatory activity of

yeast strains using the dorsal air pouch model

To determine the potential in vivo probiotic properties of

the yeast strains included in this study, we examined their

952

832

840a

982

KK

1

KK

2.1

KK

2.5

KK

3.1

KK

4.1

KK

6.5

KK

6ρ

KK

5Y3

NC

YC

9 41

4 H

. win

gei

50 60 70 80

92%

90 80

(a)

(b)

100 Strain

952 16

16

17

17

16

16

16

17

982

746

832

840a

UL

NCYC167 (R)

NCYC9 (R)

CBS1954 (R)(D)

570

630

KK2·5 (D)

KK4·1 (D)

KK2·1 (D)

KK3·1 (D)

Strain

KK5Y3

6 14 14 14 14 5

– >4

No chr

5 16 16 16 16

414

KK6·5 (D)

KK6P(D)

KK1

No chr

99%

81% 99%

II

II

I

III

I

90 100

570

630

CB

S19

54

H. w

inge

i

UL

746

1900

Kbp

1640 945 815 745 680 610 555 450 375 295 225

Mbp

3·13 2·70 2·35

1·81 1·66 1·37

1·05

0·08

NC

YC

167

Y

PH

80

YP

H80

Figure 1 Pulsed-field gel electrophoresis patterns and clustering of (a) Saccharomyces strains and (b) Candida, Isaatchenkia, Debaryomyces and

Kluyveromyces yeast strains. Similarities were estimated by the use of Pearson product moment correlation coefficient (r) and the unweighted pair

group algorithm with arithmetic averages (UPGMA). R, reference strain; D, diploid yeasts; Chr, chromosomes.

Characterization of candidate probiotic yeasts A. Kourelis et al.

264 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271

ª 2010 The Authors

capacity to induce immune reactions using the air pouch

model. The probiotic strains UL and NCFB 1748 were

used as positive controls while the strains NCYC167,

NCYC9, CBS1954 and 952 were also included as negative

controls. One-way anova analysis of the number of air

pouch PMN revealed that only the strains 982, 840a,

KK1, 570, 630 and KK6P induced the accumulation of

significantly (P < 0Æ05) high number of cells in compari-

son with the control group (Fig. 2). The probiotic strains

UL and NCFB 1748 increased the number of PMN into

the air pouch at 4 · 106 and 6Æ5 · 106 cells, respectively.

Based on the above, the 570, 630, 982 and KK1 yeast

strains whose injection into the air pouch resulted in the

accumulation of more than 4 · 106 PMN were arbitrarily

accepted as strains possessing strong immunostimulatory

activity and thus were further analysed. The above data

indicate that the immunostimulatory activity of yeast

strains in the air pouch is strain specific.

Effect of yeast strains on air pouch PMN phagocytic

activity

Probiotics have been shown to enhance the phagocytic

activity of PMN (Delcenserie et al. 2008). The data

presented in Table 2 show increased ex vivo phagocytic

activity of PMNs accumulated in the air pouch in response

to the administration of the 982, KK1, 570 and 630 strains.

At 5 min post initiation, a 40–55% phagocytosis was

detected reaching maximum at 30 min. These responses

were similar to the phagocytosis induced after administra-

tion in the air pouch of the probiotic UL strain. The 832

strain previously (Fig. 2) characterized with low immuno-

stimulatory activity and included as negative control,

exhibited significantly lower phagocytic activity than the

above strains, indicating that low PMN recruitment in the

air pouch is associated with low phagocytic activity.

All the above data show that among the yeast strains

tested, only the 982, KK1, 570 and 630 strains induced

the activation of innate immune responses in both rat

and mouse air pouch that are of comparable potency

8 # *

*

*

* *

*

*

*

#

# #

7

6

5

4

3

Num

ber

of a

ir po

uch

cells

(×

10–6

ml–1

)

2

1

0

Con

trol

84

0a 982

KK

1 57

0 63

0 K

K6P

UL

NC

YC

167

NC

YC

9

746

832

952

414

KK

2.1

KK

2.5

KK

3.1

KK

4.1

KK

6.5

KK

5Y3

CB

S19

54

NC

FB

1748

Figure 2 Determination of PMN cell recruitment in mouse air

pouches after injection of the probiotic strains UL, NCFB 1748 and

the yeast strains. Data are presented as means ± SEM (n = 5).

*Statistically significant difference in comparison with the control

group, P < 0Æ05. #Statistically nonsignificant difference in comparison

with the probiotic UL or NCFB 1748 strains. ( ) Control; ( ) strains

with increased PMN accumulation; ( ) probiotic strains and ( )

strains with low PMN accumulation.

Table 2 Determination of phagocytic activity of PMN cells and cytokine production in yeast-treated mouse air pouches

Strains

Phagocytic activity (%)�

Concentration of cytokines in mouse air pouch

exudates (pg ml)1)�

5¢ 15¢ 30¢ TNF-a IFN-c IL-10

Control 11Æ3 ± 1Æ12 30Æ0 ± 1Æ44 35Æ6 ± 0Æ98 29Æ9 ± 1Æ93 89Æ4 ± 19Æ52 43Æ7 ± 5Æ04

Saccharomyces cerevisiae

832 21Æ4 ± 0Æ44 30Æ5 ± 4Æ86 46Æ9 ± 7Æ44 72Æ5 ± 13Æ28* 95Æ8 ± 31Æ94 235Æ9 ± 37Æ23

982 49Æ9 ± 1Æ20 61Æ3 ± 2Æ69 73Æ8 ± 2Æ05 254Æ3 ± 19Æ86* 413Æ9 ± 97Æ43* 547Æ4 ± 50Æ78*

Saccharomyces boulardii

KK1 43Æ0 ± 2Æ32 58Æ6 ± 3Æ73 70Æ3 ± 0Æ96 396Æ9 ± 19Æ50* 43Æ4 ± 11Æ85 719Æ3 ± 96Æ93*

UL 40Æ1 ± 1Æ55 48Æ3 ± 0Æ46 66Æ3 ± 1Æ75

Kluyveromyces lactis

570 54Æ3 ± 6Æ44 65Æ4 ± 9Æ37 73Æ8 ± 1Æ52 92Æ8 ± 17Æ56* 47Æ9 ± 14Æ16 102Æ7 ± 44Æ45

630 51Æ2 ± 3Æ73 65Æ0 ± 2Æ06 77Æ3 ± 3Æ40 145Æ1 ± 37Æ78* 31Æ9 ± 8Æ25 124Æ5 ± 54Æ95

TNF-a, tumour necrosis factor alpha; IFN-c, gamma interferon; IL, interleukin.

*Statistically significant difference in comparison with the control group, P < 0Æ05.

�Mean percentage ± SEM of air pouch cells with phagocytosed baker’s yeasts (n = 3).

�Data are presented as means ± SEM (n = 5).

A. Kourelis et al. Characterization of candidate probiotic yeasts

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271 265

with the respective responses induced by the probiotic UL

and NCFB 1748 strains.

Determination of TNF-a, IFN-c and IL-10 in air pouch

exudates

To examine whether the above yeast strains induce

increased production of cytokines in the air pouch, we

performed ELISA immunosorbent assay in the air pouch

exudates. The data in Table 2 indicate differential ability

of the yeast strains tested to induce a specific cytokine

profile, i.e. TNF-a, IFN-c and IL-10. The 982 strain

induced increased production of TNF-a, IFN-c and IL-10;

the KK1 strain induced the production of TNF-a and

IL-10, while the strains 570 and 630 induced only TNF-aproduction (Table 3). These data show strain-specific

activation of the cells forming the air pouch that in turn

produce and secrete certain profile of cytokines.

All the above air pouch model data demonstrate that

the strains 982, KK1, 570 and 630 exhibited immunostim-

ulatory activity similar to that induced by the probiotics

UL and NCFB 1748 suggesting potential activation of the

immune responses in the gut mucosa as well.

Determination of IgA, COX-1, COX-2, cytokine and TLR

producing cells in the gut mucosa

To examine whether a selected set of yeast strains (630,

982, KK1) induce similar immune responses in the air

pouch and the intestine, they were orally administered to

mice for 10 days. We have previously shown that several

Lactobacillus strains exhibit similar immunostimulatory

activity in the small and large intestine (Kourelis et al.

2010a). However, higher responses concerning cytokine

and TLR production were observed in the small intestine.

Thus, yeast strains’ immunostimulatory activity was

determined only in the small intestine. The two K. lactis

(570, 630) strains showed similar PFGE chromosome pro-

file (Fig. 1) and exhibited similar immune effects in the

air pouch indicating that they potentially belong to the

same strain. Thus, only the 630, 982 and KK1 strains

were further analysed.

All strains increased the number of IgA producing cells

in the small intestine lamina propria with the KK1 strain

exhibiting the highest response (Fig. 3a, Table 3). This

induction occurred in the absence of inflammatory

immune response because yeast oral administration did

not increase the number of cells producing the inflamma-

tory mediators COX-1 and COX-2. In addition, there was

no evidence of any morphological alteration in the small

intestine or modification in the structure of the villi

(Fig. 3b,c).

All three yeast strains induced a significant increase in

the number of TNF-a, IL-5, IL-6 and IL-12 positive cells

(P < 0Æ05) in comparison with the control group (Fig. 4a,

Table 3). Moreover, the strains 982 and KK1 increased

the number of IL-10 producing cells, while only the strain

982 caused a significant increase in the number of IFN-cproducing cells. All cytokine producing cells were

detected in the epithelium and the lamina propria

(Fig. 4b,c). The above data show that the three yeasts

exhibit differential activation of cytokine production in

the small intestine that is similar to the response induced

by these strains in the air pouch.

To determine the signalling events leading to the

induction of the above cytokine profile, we also examined

the TLR expression in small intestine cells. Oral adminis-

tration of the yeast strains 630, 982 and KK1 resulted in a

significant increase in the number of intestinal cells

expressing TLR2 and TLR6 (Fig. 5, Table 3). In addition,

the strains 630 and 982 increased the number of TLR4-

positive cells, while the 982 strain also caused a significant

Table 3 Comparison of the immune responses induced in the air pouch and the small intestine of mice after administration of yeast strains

Strains*

Air pouch Small intestine

Chemotaxis Phagocytosis

Cytokine production IgA and cytokine production TLR expression

TNF-a IFN-c IL-10 IgA TNF-a IFN-c IL-5 IL-6 IL-10 IL-12 TLR2 TLR4 TLR6 TLR9

Saccharomyces cerevisiae

832 ) ) + ) )982 + + + + + + + + + + + + + + + +

Saccharomyces boulardii

KK1 + + + ) + + + ) + + + + + ) + )UL + +

Kluyveromyces lactis

570 + + + ) )630 + + + ) ) + + ) + + ) + + + + )

TNF-a, tumour necrosis factor alpha; IFN-c, gamma interferon; IL, interleukin; TLR, Toll-like receptors.

*The yeast strains exhibiting low chemotaxis are not presented in this table.

Characterization of candidate probiotic yeasts A. Kourelis et al.

266 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271

ª 2010 The Authors

increase in the number of TLR9-positive cells. TLR

expression was observed mainly in the crypt cells, as well

as in the epithelium and the lamina propria.

Discussion

Modulation of the innate or adaptive immunity is consid-

ered as an important property of a potential probiotic

strain. Given the recent concerns on probiotic treatment,

it is important to investigate their local and systemic

immune activity (Vogel 2008). In this study, we observed

early activation of innate immune responses such as

increased PMN recruitment and ex vivo phagocytic capac-

ity as well as cytokine production in the air pouch by

yeast strains exhibiting in vitro probiotic properties. These

early proinflammatory responses share similarities with

the responses induced by these strains in the gut mucosa

after their oral delivery. These data are in line with our

previous study showing the immunostimulatory activity

of potential probiotic Lactobacillus strains in both the air

pouch and the gut mucosa (Kourelis et al. 2010a) and

furthermore demonstrates that in addition to S. boulardii

other yeast species such as S. cerevisiae or K. lactis may

also exhibit in vivo probiotic properties. This study also

shows that the air pouch system provides the environ-

ment for a rapid and reliable discrimination of potential

probiotic yeast strains exhibiting immunostimulatory

capacity in the gut.

Characterization of a strain and its origin are very

important, as this will provide an indication of its pre-

sumed safety and meet requirements for exact informa-

tion on the nomenclature of the strain (Salminen et al.

2001). On the basis of polyphasic and reliable taxonomy,

we used Biolog YT microplate and chromosomal PFGE

karyotyping for the identification of yeasts. Our taxo-

nomic analysis using Biolog YT microplate revealed dif-

ferent identification for three of the 16 strains tested

(Table 1). In line with previous data (Praphailong et al.

1997), our results also show the potential of Biolog YT to

be used for the identification of yeasts of food and

human origin. The number and distribution of chromo-

some bands were in agreement with previous yeast PFGE

chromosome profiles (Doi et al. 1994; Belloch et al. 1998;

Keogh et al. 1998; Corredor et al. 2003). However, PFGE

alone seems to be insufficient for the characterization of

yeasts with a divergent chromosome profile such as the

D. hansenii species but in association with sensitive bio-

chemical methods such as Biolog YT microplate could be

used for the reliable identification of yeasts.

Our findings showed that from the 15 strains exhibiting

in vitro probiotic properties, only four of them possessed

immunostimulatory activity in the in vivo air pouch

(a) 50

** *

40

30

20

10

0N

umbe

r of

pos

itive

cel

ls p

er 1

0 fie

lds

IgA COX-1 COX-2

(b) (c)

Figure 3 Effect on the initiation of inflamma-

tory or immune responses in the intestinal

mucosa of mice fed with the assayed yeast

strains (a) Quantitative analysis of IgA,

cyclooxygenase (COX)-1 and COX-2

producing cells in the small intestine after oral

administration of 630, 982 and KK1 yeast

strains. Data are presented as means ± SEM

(n = 5). *Statistically significant difference in

comparison with the control group, P < 0Æ05.

(b) Light microphotograph of haematoxylin–

eosin-stained sections from the small intestine

of unfed mice. (c) Light microphotograph of

haematoxylin–eosin-stained sections from the

small intestine of mice fed with the assayed

strains. ( ) Control; ( ) 630; ( ) 982 and ( )

KK1.

A. Kourelis et al. Characterization of candidate probiotic yeasts

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271 267

model. The air pouch provides an excellent system to dis-

criminate presumptive probiotic yeast strains, because it

shows similar immune responses with the intestine and

outweighs the in vitro assays such as cell culture experi-

ments used for probiotic characterization. In addition, the

potential immunostimulatory activity in the air pouch

could be determined within 3 h after a single injection of

yeast cells and outweighs the delivery in the intestinal tract

that requires daily administration for at least 7 days. We

have demonstrated the K. lactis 570 and 630 strains, the

S. cerevisiae 982 strain and the S. boulardii KK1 strain

exhibit in the air pouch increased PMN influx, phagocytic

ability and cytokine production of comparable potency

with the probiotics UL and NCFB 1748 (Fig. 1, Table 2,

Kourelis et al. 2010a). Strains negative for in vitro probiot-

ic properties such as 952 and strains positive for in vitro

probiotic properties such as 832 were used as low

responders concerning PMN recruitment and phagocytosis

suggesting potential strain-specific regulation of immune

proinflammatory reactions in the air pouch.

Probiotic yeast strains of the species S. boulardii have

been shown to increase the phagocytic activity and cyto-

kine production (Rodrigues et al. 2000; Czerucka et al.

2007). The strains 570, 630, 982 and KK1 can positively

influence innate immune responses, indicating that these

strains may exhibit pathogen clearance activity. Although

the dorsal air pouch does not simulate the intestinal func-

tion, the air pouch lining tissue and the intestine mucosa

share similarities as they both exhibit a barrier function

that is achieved by epithelial cells and resident macro-

phages (Sedgwick et al. 1983; Wittig and Zeitz 2003;

Magalhaes et al. 2007). Furthermore, the above yeast

(b) (c)

120(a)

100

80

60

**

*

* * * *

* *

*

*

* * * *

40

20

0TNF-a IFN-g IL-5 IL-6 IL-10 IL-12B

Num

ber

of p

ositi

ve c

ells

per

10

field

s

Figure 4 Effect of the assayed yeast strains

on the cytokine profile of mice small intestine

(a) Quantitative analysis of tumour necrosis fa-

ctor alpha (TNF-a), gamma interferon (IFN-c),

interleukin (IL)-5, IL-6, IL-10 and IL-12 produc-

ing cells after oral administration of 630, 982

and KK1 yeast strains. Data are presented as

means ± SEM (n = 5). *Statistically significant

difference in comparison with the control gro-

up, P < 0Æ05. (b) Microphotography of IL-6-p-

ositive cells in the epithelium and lamina

propria of the small intestine of (control) unfe-

d mice. (c) Microphotography of cytokine-pos-

itive cells in the epithelium and lamina propria

of the small intestine of mice fed with the ass-

ayed strains. ( ) Control; ( ) 630; ( ) 982 a-

nd ( ) KK1.

80

70

60

50

*

*

*

*

*

*

*

*

*

40

30

20

10

0TLR2 TLR4 TLR6 TLR9

Num

ber

of p

ositi

ve c

ells

per

10

field

s

Figure 5 Quantitative analysis of Toll-like receptor (TLR)2, TLR4, TLR6

and TLR9 producing cells in the small intestine after oral administra-

tion of 630, 982 and KK1 yeast strains. Data are presented as

means ± SEM (n = 5). *Statistically significant difference in compari-

son with the control group, P < 0Æ05. ( ) Control; ( ) 630; ( ) 982

and ( ) KK1.

Characterization of candidate probiotic yeasts A. Kourelis et al.

268 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 260–271

ª 2010 The Authors

strains interact with the cells forming the air pouch lining

tissue and the gut-associated lymphoid tissue (GALT)

with subsequent increase in cytokine production. In addi-

tion, the cytokine profile of the 630, 982 and KK1 strains

in the air pouch was similar to the cytokine profile

observed in the small intestine after oral administration

of these strains (Table 3). Thus, the interactivity of the

yeast strains with the air pouch may be the signal initiat-

ing the proinflammatory response by the production of

species-specific cytokines. TNF-a is necessary to initiate

the crosstalk between the immune cells of the lamina pro-

pria and the intestine epithelial cells (Galdeano et al.

2007). The increase in TNF-a in the air pouch could sug-

gest that TNF-a may be required to initiate the interac-

tion among the epithelial cells, the PMN cells and the

resident macrophages of the air pouch membrane. The

precise mechanism by which 570, 630, 982 and KK1 exert

immunostimulatory activity in the air pouch remains

unclear in this study. A possible mechanism could involve

the rapid activation of TLRs (Doyle et al. 2004), the com-

plement cascade and the production of chemokines (Yam

et al. 2008). These chemotactic factors increase the

recruitment of PMNs exhibiting strong phagocytic activity

(Coates and McColl 2001) and the subsequent production

of cytokines such as TNF-a, IFN-c (Diamond et al. 1991;

Utsunomiya et al. 1998).

The interaction of yeast strains with the GALT is not

followed by the activation of an inflammatory response

because their oral delivery does not result in morphologi-

cal changes of the villi, in massive accumulation of PMNs

or in increased number of COX-1 and COX-2 producing

cells (Fig. 3). These differences in the small intestine vs

the air pouch may be attributed to the higher number of

resident immune cells and yeast antigen availability in the

air pouch than in the intestine.

Oral administration of 630, 982 and KK1 strains caused

a significant increase in the number of IgA producing

cells. Such a response has been previously reported for

the probiotic S. boulardii species (Rodrigues et al. 2000).

Increased IgA production suggests a potential role of

these strains in the immunity against invading pathogenic

microorganisms and the maintenance of intestinal

homeostasis (Fagarasan and Honjo 2004).

Interaction of yeast strains with the gut mucosa acti-

vated a strain-specific profile of cytokines and TLRs

(Fig. 4, Table 3). The strain 630 induced the production

of TNF-a, IL-5, IL-6 and IL-12 in a TLR2, -4, -6 depen-

dent manner indicating no bias towards Th1 or Th2 ⁄ Treg

intestinal immune response. Furthermore, the increased

number of IL-5 and IL-6 producing cells could also con-

tribute in the maintenance of tolerance as well as IgA

switch and secretion in the gut mucosa (Corthesy et al.

2007). The strain 982 induced the production of TNF-a,

IFN-c, IL-5, IL-6, IL-10 and IL-12 through TLR2, -4, -6,

-9. The cytokine profile of increased levels of both IL-12

and IFN-c by the 982 strain indicates that this strain may

favour the induction of a Th1 response. The strain KK1

activated the profile of TNF-a, IL-5, IL-6, IL-10 and

IL-12 through TLR2, -6. The KK1 strain may favour the

induction of a Th2 ⁄ Treg response as a result of the pro-

duction of IL-5 and IL-10. The Saccharomyces strains

(982 and KK1) may also exert a potent anti-inflammatory

activity in the intestine through the production of IL-10

that would prevent Th1-driven local and systemic inflam-

matory responses (Rennick et al. 1992). Oral administra-

tion of the KK1 strain in gluten-sensitive mice inhibited

the induction of gluten-induced enteropathy that is

characterized by small intestine inflammatory lesions and

specific Th1 responses (V. Gerakopoulos unpublished

data). Activation of combinations of certain TLRs by the

three yeast strains is required for the high discriminatory

ability of TLRs (Roeder et al. 2004). Our findings suggest

that the immunostimulatory activity of the yeast strains

examined is mediated through the interaction of TLR4

with the cell wall glucan present on the strains 630 and

982, or through TLR2, -6 heterodimer interaction with

mannan present on the strains 630, 982 and KK1 (Roeder

et al. 2004). In addition, mannan or zymosan CpG–DNA

complexes recognized by TLR9 (Anada et al. 2006; Dan

et al. 2008) may also be responsible for the immuno-

stimulatory activity of the 982 strain.

In conclusion, a combination of phenotypic, biochemi-

cal and genotypic assays should be used for the identifica-

tion of yeasts. The strains S. cerevisiae 982, S. boulardii

KK1 and K. lactis 630 could be considered as candidate

probiotics, further supporting the use of other yeast

strains besides S. boulardii as probiotics. Furthermore, the

dorsal air pouch model may be used to rapidly and effi-

ciently discriminate among presumptive probiotic yeast

strains with immunostimulatory activity. Under normal

conditions, this stimulation of the host’s mucosal immune

system by nonpathogenic microorganisms, through a

highly regulated system of TLR and cytokine expression,

may have favourable effects on the development and

maintenance of the immune system and may provide

the host with a higher capacity to resist any inflamma-

tory response. Finally, further experimentation is

required using disease animal models to confirm the

immunomodulatory activity of the above yeast strains

while final validation of the findings of this study must

await clinical trials.

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