ORIGINAL ARTICLE

Validation of the dorsal air pouch model to predict andexamine immunostimulatory responses in the gutA. Kourelis1, I. Zinonos1, M. Kakagianni1, A. Christidou1, N. Christoglou1, E. Yiannaki2, T. Testa1,C. Kotzamanidis1, E. Litopoulou-Tzanetaki3, N. Tzanetakis3 and M. Yiangou1

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

2 Haematological Laboratory, Anticancer Hospital ‘‘Theagenio’’, Thessaloniki, Greece

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

Introduction

Several nonpathogenic bacteria are given as probiotics

or as live bacterial vaccines or as gene delivery vectors

(Steidler et al. 1998; Steidler 2003; Braat et al. 2006).

Given the recent concerns on probiotic treatment, it is

important to investigate their local and systemic

immune activities (Vogel 2008). Probiotics are defined

as ‘live micro-organisms that when being administered

in adequate amounts confer a health benefit on the

host’ (FAO ⁄ WHO 2002). The health benefits of probio-

tics are achieved by bacterial antagonism and immuno-

modulation, which help the healthy host to maintain a

‘physiological state of inflammation’ in the intestine

(Galdeano et al. 2007) or to control several infectious,

inflammatory and immunologic reactions. The pene-

tration of probiotics or their antigens in the intestine

activates the innate immunity through interaction with

the Toll-like receptors (TLRs) present on immune cells

(Akira et al. 2001) and subsequently the production of

cytokines and chemokines (Vinderola et al. 2005).

Under physiological conditions, probiotic bacteria can

act also as mucosal and systemic adjuvants (Galdeano

et al. 2007).

Keywords

air pouch, cytokines, gut mucosa,

Lactobacillus paracasei, probiotic, toll-like

receptors.

Correspondence

Minas Yiangou, Department of Genetics,

Development and Molecular Biology, Biology

School, Aristotle University of Thessaloniki,

54124 Thessaloniki, Greece.

E-mail: Yiangou@bio.auth.gr

2009 ⁄ 0023: received 6 January 2009, revised

7 May 2009 and accepted 21 May 2009

doi:10.1111/j.1365-2672.2009.04421.x

Abstract

Aims: To validate the use of the air pouch system to predict and examine early

immune responses induced by the presumptive probiotics Lactobacillus paraca-

sei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut

mucosa.

Methods and Results: Only the DC412 strain interacted strongly with the cells

forming the air pouch lining tissue and induced early innate immune responses

such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour

necrosis factor alpha (TNF-a) production that equal the respective responses

induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains

exhibiting strong immunoregulatory activity in the air pouch also interacted

strongly with the gut-associated lymphoid tissue (GALT). The strain DC412

exerts its effect on the intestine through stimulation of Toll-like receptor

(TLR)2 ⁄ TLR4-mediated signalling events leading to secretion of a certain pro-

file of cytokines in which gamma interferon (IFN-c), TNF-a, interleukin (IL)-6

and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the

same cytokine profile in addition to IL-12B, and this response is potentially

mediated by the synergy of TLR2 and TLR9.

Conclusion: The strain DC412 possesses the in vitro and in vivo characteristics

of a probiotic micro-organism.

Significance and Impact of the Study: The dorsal mouse or rat air pouch may

be used as an alternative and rapid method for the initial discrimination and

selection of potential probiotic Lactobacillus strains.

Journal of Applied Microbiology ISSN 1364-5072

274 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284

ª 2009 The Authors

Mucosal and systemic administration of probiotics

promotes IgA antibody production (Vitini et al. 2000;

Alvarez-Olmos and Oberhelman 2001), and the induction

of a certain cytokine pattern profile that in turn regulates

the helper T cell 1 (Th1) ⁄ helper T cell 2 (Th2) balance

(Galdeano et al. 2007). The positive influence of many

probiotics on the phagocytic capacity of monocytes and

polymorphonuclear (PMN) cells has been recently

reviewed (Delcenserie et al. 2008). Macrophages and

PMN represent the first line of host defense against

pathogens, and probiotic-mediated local increase in PMN

recruitment successfully treated bovine mastitis (Crispie

et al. 2008). The route of probiotic delivery and the sub-

sequent PMN recruitment and phagocytic activity as well

as chemokine and cytokine production at the local site of

administration are early events and important parameters

for the initiation of non-well-defined immune responses.

In previous studies, we isolated several Lactobacillus

paracasei subsp. paracasei strains from infant’s gastroin-

testinal tract (faeces) or feta cheese prine (Tzanetakis and

Litopoulou-Tzanetaki 1992; Xanthopoulos et al. 2000).

These strains possessed biotechnologically important

characteristics (Litopoulou-Tzanetaki and Tzanetakis,

1992) as well as in vitro probiotic properties such as

viability at low pH and in human gastric juice, bile salt

tolerance, antibacterial activity and cholesterol reduction

(Xanthopoulos et al. 1998, 2000). These observations

provide us with the biological system to study the mech-

anism of the early local action of Lactobacillus strains

possessing in vitro probiotic properties on immune

responses. The 6-day-old epithelium-enclosed air pouch

is developed by epithelial, phagocytic and fibroblast-like

lining cells and comprises the intima (epithelial and poly-

morphonuclear cells) and the subintima (macrophages

and fibroblasts) layers, with an abundance of cells fol-

lowed by an organized vasculature that acts as a mechan-

ical barrier (Sedgwick et al. 1985; Coates and McColl

2001). Activation of the air pouch by antigen causes the

rapid recruitment of PMN cells, while the immune

response products such as cytokines and chemokines are

retained in the air pouch, enhancing early inflammatory

responses not easily detectable in systemic immunity.

Bacterial administration in the air pouch involves their

interaction with the barrier-forming resident macrophag-

es and epithelial cells (Yam et al. 2008). Importantly,

probiotics or their antigens interact with the intestine

barrier through the epithelia of gut mucosa and dendritic

cells and macrophages of villi and Peyer’s patches (Perdi-

gon et al. 2001; Dogi and Perdigon 2006). The similarity

of bacterial activation between the intestine and the air

pouch barriers indicates that the dorsal air pouch may

provide an excellent model to discriminate presump-

tive probiotic strains for their immunostimulatory activ-

ity by examination of their early local effects on immune

responses such as cell-to-cell interactions, chemotaxis and

phagocytic activity. We present data showing that the

Lact. paracasei DC412 strain as well as the Lactobacillus

acidophilus NCFB 1748 probiotic strain exhibit immuno-

regulatory activity in both the dorsal air pouch and the

gut mucosa.

Materials and methods

Bacterial strains and growth conditions

The Lact. paracasei subsp. paracasei B112, DC205, DC215

(Tzanetakis and Litopoulou-Tzanetaki 1992) and DC412

(Xanthopoulos et al. 2000) strains were obtained from the

collection of the Laboratory of Food Microbiology and

Hygiene, Aristotle University of Thessaloniki. The strains

DC205 and DC215 may be considered as possible probiot-

ics because of their ability to withstand low pH values

(pH 3Æ0) and high bile concentration (N. Tzanetakis and

E. Litopoulou-Tzanetaki; unpublished data). The probiotic

strain Lact. acidophilus NCFB 1748 was isolated from

human intestine (Efthymiou and Hansen 1962) and is

commonly used by dairy industry as probiotic (Sullivan

and Nord 2006). NCFB 1748 was included in this study

on the basis that exhibited in vitro probiotic properties

similar to those described for Lact. paracasei strains

(Xanthopoulos et al. 1998, 2000) as well as immunomod-

ulatory activity (Nerstedt et al. 2007). The Lactobacillus

helveticus ATCC 15009 strain was commercially available

and exhibited no proven in vitro or in vivo probiotic prop-

erties. All lactobacilli were stored at )80�C in MRS broth

with 25% glycerol. Lactobacilli were subcultured at least

three times prior to experimental use. NCFB 1748 and

ATCC 15009 strains were grown for 18 h at 37�C, while

all Lact. paracasei subsp. paracasei strains were grown for

18 h at 30�C.

Preparation of strains

Bacterial cells from an overnight culture were washed

twice with 0Æ9% NaCl and finally resuspended at the

appropriate number (CFU ml)1) according to McFarland

standard. Labelling of Lactobacillus strains or heat-killed

baker’s yeast (Saccharomyces cerevisiae) with fluorosceine

isothiocyanate (FITC) was performed as described previ-

ously (Dogi and Perdigon 2006). Briefly, cells were washed

twice with phosphate-buffered saline (PBS) and then incu-

bated for 1 h at 37�C in 0Æ05 mol l)1 Na2CO3, pH 9Æ0,

containing 100 lg ml)1 FITC. After repeated washings,

the Lactobacillus-FITC or yeast-FITC pellets were resus-

pended in saline at 5 · 108 CFU ml)1 or 107 cells ml)1,

respectively, and stored at 4�C until used.

A. Kourelis et al. Probiotic injection in mouse air pouch

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284 275

Animals

Balb ⁄ c (20–30 g) inbred mice or Fisher-344 inbred rats

(130–180 g) were housed under standard laboratory con-

ditions (12 h light ⁄ 12 h dark cycle) and received a diet of

commercial food pellets and tap water. Animals were

sacrificed by light ether anaesthesia followed by cervical

dislocation. All experiments were performed in an accred-

ited animal facility (number EL 54 BIO 02, School of

Biology, Aristotle University of Thessaloniki) and com-

plied with the current ethical regulations on animal

research of our university, which is according to both

Greek National and EU legislation. All groups included in

the present study consisted of five animals, and each

experiment was repeated at least two times.

Formation of the air pouch

Air pouches were created on the back of mice or rats as

described previously (Sedgwick et al. 1985) by subcutaneous

injection of 3 or 20 ml sterile air, respectively. The forma-

tion of equal size of air pouch in each animal was achieved

by refilling the 3- and 5-day-old air pouches with the

appropriate volume of air and confirmed using a vernier.

Subsequently, Lactobacillus strains were administered in the

6-day-old mouse or rat air pouches. Mice were injected with

0Æ2 ml pyrogenic-free saline containing 108 CFU of bacteria,

while rat air pouches received 5 · 108 CFU of bacteria in

1 ml of pyrogenic-free saline. Control animals in the air

pouch received only pyrogenic-free saline.

Determination of PMN accumulation in Lactobacillus

treated air pouches

The PMN cells accumulated in Lactobacillus-treated rat or

mouse air pouches were harvested 3 h postbacteria adminis-

tration after injection in the air pouch of 10 or 2 ml of saline,

respectively, followed by air pouch lavage. This time point

was selected because time-course kinetic experiments

revealed that in air pouches injected with the NCFB 1748

strain, the maximum accumulation of PMN cells and comp-

lete bacterial clearance occurred 3–6 h postbacteria admini-

stration (data not shown). After centrifugation, 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 of air pouch PMN cells

Phagocytic activity of the rat air pouch PMN cells was

determined as described previously (Loose et al. 1978).

Heat-killed baker’s yeasts (106 cells) were opsonized by

incubation with rat serum for 30 min at 37�C. Then, the

106 opsonized heat-killed baker’s yeasts were mixed with

2Æ5 · 106 air pouch PMN cells and incubated at 37�C for

additional 30 min. Phagocytosis was stopped by the addi-

tion of FBS. Samples were then mixed with trypan blue,

and PMN cells containing phagocytosed yeasts were

counted using a haemocytometer under light microscope.

Phagocytic activity of air pouch PMN cells was also deter-

mined by flow cytometric-based assay using FITC-labelled

baker’s yeast (Nuutila and Lilius 2005). Samples were

then analysed on an EPICS XL (Coulter, Miami, FL,

USA) cytometer. At least 3 · 106 events were measured

for each sample at a low flow rate. To exclude yeast

adhesion from phagocytosis, parallel experiments were

performed on ice (0–4�C) with or without the addition

of trypan blue to quench external fluorescence. The

percentage of fluorescence of air pouch PMN cell popu-

lation on ice was excluded from the percentage estimated

for the same population at 37�C.

Cytokine detection in air pouch exudates

Air pouch exudates isolated from control or Lactobacillus

treated air pouches were subjected to ELISA to detect the

levels of gamma interferon (IFN-c), tumour necrosis

factor alpha (TNF-a) and interleukin (IL)-10 using the

ELISA kit (eBioscience, Inc., San Diego, CA, USA),

according to manufacturer’s instructions. As positive sam-

ples for the assay, we used purified cytokines included in

the kit as well as air pouch exudates isolated from bacte-

rial lipopolysaccharide or Freund’s complete adjuvant-

treated air pouches.

Preparation of paraffin blocks

Mouse air pouch membrane or intestine was removed,

dissected into small pieces, fixed in 4% paraformaldehyde

in PBS and finally embedded in paraffin blocks (Sainte-

Marie 1962). Sections of 4 lm thickness were either

stained with haematoxylin–eosin or further processed for

immunohistochemistry (Avramidis et al. 2002).

Interaction of Lactobacillus strains with air pouch lining

tissue or gut-associated lymphoid tissue (GALT)

Mice injected in the air pouch with 200 ll of each FITC-

labelled Lactobacillus strain (108 CFU) or orally received

100 ll of each FITC-labelled bacterial strain at doses of

108 CFU ml)1 were sacrificed 3 h or 20 min later, respec-

tively. To investigate whether the administered strains

interact with the air pouch lining tissue or GALT, air

pouch membranes and small or large intestine were

Probiotic injection in mouse air pouch A. Kourelis et al.

276 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284

ª 2009 The Authors

removed and embedded in paraffin blocks as described

above.

Immunohistochemical detection of cells producing IgA,

cytokine and TLR in the small and large intestines

Mice housed in separate cages orally received 50 ll saline

containing 109 CFU of each of the bacterial strains for

ten consecutive days, while control mice received only

50 ll of pyrogenic-free saline. One day after the last dose,

the intestine was removed and processed for paraffin

embedding.

The number of cells producing IgA or TLR2 was deter-

mined on samples by direct immunofluorescence using

monospecific anti-IgA (Sigma, St Louis, MO, USA) anti-

body or TLR2 (Santa Cruz Biotechnology, Inc., Santa

Cruz, CA, USA) conjugated with FITC. Indirect immuno-

fluorescence was used to determine the cells producing

TLRs 4, 6 and 9, cytokines and inflammatory mediators

by using specific antibodies directed against mouse TLRs

4, 9 (Abcam plc, Cambridge, UK), TLR6, COX-1, COX-2,

IL-12B (Santa Cruz Biotechnology, Inc), IFN-c, TNF-aand IL-10 (R&D systems, Inc., Minneapolis, MN, USA).

Antibodies were applied for 2 h at room temperature,

and after four washes in PBS, the slices were incubated

for 1 h with the FITC-conjugated anti-goat IgG (Sigma)

as secondary antibody. After four washes in PBS, the sam-

ples were examined using fluorescence light microscope.

The number of positive cells was counted in ten fields at

400· magnification. Samples incubated only with the

secondary antibody were also included to confirm

specificity of the immunodetection, and the number of

positive cells was excluded from the number of cells esti-

mated for testing samples. For the final scoring, two

investigators achieved consensus.

Statistical analysis

The results are reported as the mean ± SEM. Multiple

comparisons were performed by one-way anova followed

by Tukey’s test, and statistical significance was accepted at

values of P < 0Æ05.

Results

Recruitment of PMN in Lactobacillus-treated air pouches

To determine the in vivo probiotic characteristics of the

four Lact. paracasei subsp. paracasei strains included in

this study, we examined their ability to activate immune

responses in the dorsal air pouch of mice. The strains

Lact. acidophilus NCFB 1748 and Lact. helveticus ATCC

15009 were used as positive and negative controls, respec-

tively. Administration of bacterial strains in the mouse air

pouch resulted in rapid accumulation of significant num-

ber of PMN cells in the air pouch exudates (Table 1).

Recruitment of PMN cells in the air pouch exudates was

increased by 10- to 12-fold after administration of the

strains DC412 or NCFB 1748 and by three to fivefold

after administration of the strains DC215, DC205, B112

and ATCC 15009 (Table 1). Similar data were observed

after administration of the above strains in rat air

pouches (Table 1). The above data indicate that the

DC412 strain exhibits immunostimulatory activity in both

Table 1 Determination of the immunostimulatory activity of Lactobacillus paracasei subsp. paracasei strains in the mouse and rat dorsal

air pouch

PMN recruitment in air pouch

(cells · 10)6) Cytokine levels in mouse air pouch exudates (pg ml)1)

Mouse Rat IFN-c TNF-a IL-10

Control 0Æ6 ± 0Æ1 8Æ5 ± 1Æ9 89Æ4 ± 19Æ5 29Æ9 ± 1Æ9 43Æ6 ± 5Æ1

Lactobacillus acidophilus

NCFB 1748 7Æ2 ± 1Æ1* 73Æ8 ± 8Æ7* 68Æ4 ± 14Æ8 198Æ2 ± 18Æ1* 54Æ6 ± 10Æ9

Lact. paracasei subsp. paracasei

B112 1Æ5 ± 0Æ6*� ND 58Æ3 ± 20Æ3 231Æ1 ± 40Æ3* 35Æ4 ± 12Æ6

DC205 3Æ2 ± 0Æ3*� ND 74Æ3 ± 10Æ7 94Æ91 ± 19Æ6* 41Æ2 ± 14Æ8

DC215 1Æ7 ± 0Æ5*� 33Æ7 ± 8Æ2*� 81Æ4 ± 9Æ8 135Æ7 ± 26Æ1* 44Æ3 ± 18Æ6

DC412 6Æ6 ± 0Æ8* 75Æ4 ± 7Æ6* 68Æ4 ± 14Æ8 284Æ1 ± 45Æ4* 48Æ1 ± 5Æ6

Lactobacillus helveticus

ATCC 15009 1Æ8 ± 0Æ9*� 41Æ8 ± 7Æ1*� 64Æ4 ± 12Æ7 39Æ5 ± 6Æ6 61Æ7 ± 21Æ2

Values are means ± SEM from three independent experiments (n = 5).

ND, not determined.

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

�Statistically significant differences in comparison with the probiotic NCFB 1748-treated group, P < 0Æ05.

A. Kourelis et al. Probiotic injection in mouse air pouch

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284 277

mice and rat air pouches, which equals the respective one

observed with the probiotic NCFB 1748 strain.

Effect of Lactobacillus on air pouch PMN

phagocytic activity

The total number of PMN cells accumulated in the air

pouches of normal treated mice (Table 1) is below the

number of cells (2Æ5 · 106) required to perform the phago-

cytic activity assay. To avoid mixing air pouch PMN cells

from a high number of normal mice, phagocytic activity

was performed using rat air pouch PMN cells. Kinetic

experiments to determine the phagocytic activity (Fig. 1a)

of PMN cells accumulated in air pouch exudates 3 h post-

bacteria administration by light microscopy revealed that

only the DC412 and NCFB 1748 strains induced strong

phagocytic activity. At 5-min postinitiation, 40% phago-

cytosis was detected reaching maximum at 30 min. The

strains B112, DC205 and DC215 induced significantly

lower phagocytic activity than DC412 and NCFB 1748,

indicating that low PMN recruitment in the air pouch is

associated with low phagocytic activity. Similar percentage

of phagocytosis was achieved using PMN cells isolated

from mice air pouches treated with DC412 or NCFB 1748

(data not shown). The above data were also confirmed by

FACS analysis (Fig. 1b), showing that PMN cells accumu-

lated in the dorsal rat air pouch exudates in response to

NCFB 1748 or DC412 strains administration exhibited

increased phagocytic activity of FITC-labelled yeast com-

pared to PMN cells isolated from control nontreated air

pouches or air pouches treated with DC205 or ATCC

15009 strains.

All the above data indicate that among the Lact. para-

casei strains tested, only the DC412 strain induced the

activation of innate immune responses in both rat and

mouse air pouch, which are of comparable potency with

the respective responses induced by the probiotic NCFB

1748 strain.

100(a)

(b)

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10 15 30

Time (min)

NCFB 1748

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Phagocytosis31%

Phagocytosis13·8%

Phagocytosis16·2%

1000 0·1 FITC

Fluorescence intensity

Rel

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r

1000 0·1 FITC 1000 0·1 FITC

0 min

15 min

1000

0·1 FITC 10000·1 FITC 10000·1 FITC 10000·1 FITC 1000

DC412 DC205 ATCC 15009

Figure 1 Determination of the ex vivo

phagocytic capacity of PMN cells accumulated

in rat dorsal air pouch by 3 h postlactobacilli

injection. (a) Percentage of air pouch PMN

cells showing phagocytic activity determined

by light microscopy. Each value represents the

means ± SEM of three independent experi-

ments. *Statistically significant differences in

comparison with the control group, P < 0Æ05.

(b) Flow cytometric analysis of phagocytosis

of FITC-labelled baker’s yeast by air pouch

PMN cells. ( ) Normal; ( ) NCFB 1748; ( )

DC412; ( ) DC205 and ( ) ATCC 15009.

Probiotic injection in mouse air pouch A. Kourelis et al.

278 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284

ª 2009 The Authors

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

exudates

To examine whether the above Lactobacillus strains

induce differential production of cytokines in the air

pouch, we performed ELISA immunosorbent assay in the

air pouch exudates. The data in Table 1 show that among

the cytokines tested in Lactobacillus-treated mouse air

pouch exudates, only TNF-a levels were increased signifi-

cantly by 3 h postbacteria administration. However,

DC412 as well as B112 strains induced the higher TNF-alevels followed by NCFB 1748, DC205 and ATCC 15009.

The IFN-c and IL-10 levels were not increased by any of

the lactobacilli investigated. Similar data were obtained in

Lactobacillus-treated rat air pouch exudates (data not

shown). Taken together, all the above data indicate that

the strain DC412 exhibits immunoregulatory activity that

equals the respective one observed with the probiotic

NCFB 1748.

Interaction of FITC-labelled Lactobacillus strains with

the air pouch lining tissue and the GALT

The strains DC412, DC205, NCFB 1748 and ATCC 15009

were further studied for their ability to interact with the

cells forming the intima or subintima layer of the air

pouch (Fig. 2a). By fluorescence microscopy, we observed

differential interaction of the strains NCFB 1748, DC412,

DC205 and ATCC 15009 with the air pouch lining tissue

(Fig. 2a). The FITC-labelled DC412 cells or their antigens

were associated with both the intima and subintima layers

of the air pouch (Fig. 2b), while FITC-labelled NCFB

1748 cells or antigens were detected mainly in cells form-

ing the air pouch subintima layer (Fig. 2c). The strains

DC205 and ATCC 15009 showed low interaction with the

air pouch lining tissue. The above data indicate that the

potency of Lactobacillus to interact with the air pouch is

strain specific, while the differential interaction of DC412

and NCFB 1748 strains with the cells forming the air

pouch is suggestive of possible differential activation of

immune responses by these Lactobacillus strains.

To further investigate the capacity of DC412, NCFB

1748 and DC205 strains to interact with the gut, we used

FITC-labelled bacteria. DC412 and NCFB 1748 FITC-

labelled strains and their antigens exhibited increased

capacity to interact with the GALT (Fig. 2d–f). We

observed antigens of both fluorescent bacteria in immune

cells associated with the lamina propria of the small intes-

tine, in Peyer’s patches in macrophage and DC harbour-

ing areas and in nodules and crypts of the large intestine.

The strain DC205 showed low interaction with the GALT.

The above data show that the strain-specific interaction

observed in the intestine barrier is similar with the

respective one in the air pouch. In addition, the DC412

strain interacts with the intestine cells, suggesting poten-

tial colonization ability and capacity to activate immune

responses in the gut.

Detection of cells producing IgA, COX-1 and COX-2 in

the intestine

To confirm that the DC412 strain induces immune

responses in the small and large intestines, in vivo studies

were performed by feeding mice for 10 days with the

DC412 or NCFB 1748 strains. DC205 strain was also

included as a low responder positive control. Oral admin-

istration of Lactobacillus strains in mice and subsequent

haematoxylin–eosin-stained sections of the small intestine

(a) (b) (c)

(d) (e) (f)

Figure 2 Histological analysis of FITC-labelled

lactobacilli interaction with the cells forming

the air pouch lining tissue or the gut-associ-

ated lymphoid tissue. (a) Eosin–hematoxylin

staining of normal air pouch lining tissue

showing the intima (arrowheads) and

subintima (arrows) regions. (b–c) Fluorescence

microscopy of air pouch membrane 3 h post

(b) Lactobacillus paracasei subsp. paracasei or

(c) Lactobacillus acidophilus NCFB 1748

strains injection. (d–f) Fluorescence micro-

photography showing the interaction of

FITC-labelled DC412 bacteria with (d) the

lamina propria of the small intestine, (e) the

Peyer’s patches and (f) the crypts of the large

intestine.

A. Kourelis et al. Probiotic injection in mouse air pouch

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284 279

showed no evidence of inflammatory immune response

or modification in the structure of small intestine villi

(Fig. 3a–c). Furthermore, none of the strains affected the

number of the small intestine cells that produce the

inflammatory mediators COX-1 or COX-2 (Fig. 3d).

Immunofluorescence microscopy revealed that all strains

significantly increased the number of cells producing IgA

in the lamina propria of the small or large intestine

(Fig. 3d) in comparison with the control nontreated mice.

The probiotic NCFB 1748 and the DC412 strains induced

significantly higher number of cells producing IgA than

the DC205 strain. These findings indicate that the DC412

strain activates immune responses such as IgA production

in the intestine in the absence of inflammatory response.

Detection of cells producing IFN-c, IL-10, IL-12, TNF-a

and TLRs 2, 4, 6, 9 in the intestine

The administration of both DC412 and NCFB 1748

strains increased the number of cells producing IFN-c,

TNF-a and IL-10 in the lamina propria of the small intes-

tine, while DC205 responded poorly (Fig. 4). However,

only NCFB 1748 significantly increased the number of

IL-12B positive cells in the small intestine. Similar data

were obtained in the large intestine with the exception of

the low TNF-a response for all the three strains tested

(Fig. 4a,d).

Molecular patterns of DC412, NCFB 1748 and DC205

strains significantly increased the number of cells express-

ing TLR2 and TLR4 in the small and large intestines

(Fig. 5a). In addition, NCFB 1748 strain administration

increased the expression of TLR9 mainly by the Paneth

epithelial cells located at the base of the small intestinal

crypts (Fig. 5b–c).

Discussion

The data presented in this study show that certain Lacto-

bacillus strains initiate local innate immune responses in

the dorsal air pouch as indicated by a rapid increase in

the PMN recruitment and their ex vivo phagocytic capa-

city as well as TNF-a production early post administra-

tion. These results represent the first report showing such

immunostimulatory activity in this model by Lactobacillus

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field

s

40

20

0IgA

Smallintestine

IgALarge

intestineSmall

intestine

COX-2COX-1

*

*

*

#

*

(a) (b)

(d)

(c)

Figure 3 Effect on the structure of intestine

and the initiation of inflammatory or immune

responses in the intestinal mucosa of mice fed

with lactobacilli. (a–c) Light microphotographs

of haematoxylin–eosin-stained sections from

the small intestine of mice fed for 10 days

with (a) DC412, (b) NCFB 1748 and (c) DC205

strains. (d) Effect of oral administration of

lactobacilli strains on cells producing IgA,

COX-1 and COX-2 in the small or large

intestine of mice by immunofluorescence.

The histogram is representative of two similar

experiments, and the bars represent the

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

differences in comparison with the control

group, P < 0Æ05. #Statistically significant

differences in comparison with the probiotic

NCFB 1748-treated group, P < 0Æ05. ( )

Normal; ( ) NCFB 1748; ( ) DC412 and

( ) DC205).

Probiotic injection in mouse air pouch A. Kourelis et al.

280 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284

ª 2009 The Authors

strains exhibiting in vitro probiotic properties. We pro-

pose that these early proinflammatory responses in the air

pouch share similarities with the early responses induced

by these strains in the intestine after their oral delivery.

We have demonstrated that one of the four Lact. para-

casei strains examined, the DC412 strain, exhibits in the

air pouch increased PMN influx, phagocytic capacity and

TNF-a production of comparable potency with the pro-

biotic Lact. acidophilus NCFB 1748 strain. The Lact. helv-

eticus ATCC 15009 strain that does not exhibit any

in vitro probiotic properties was a low responder concern-

ing interaction with the cells forming the air pouch bar-

rier, PMN recruitment and phagocytic activity suggesting

potential strain-specific immunoregulation in the air

pouch. Despite that the dorsal air pouch system does not

simulate the intestinal function, the air pouch lining tis-

sue and the intestine mucosa share similarities. Both the

air pouch and the GALT exhibit a barrier function that is

achieved by epithelial cells and resident macrophages. The

data presented in this study also indicate that the Lacto-

bacillus strains initiate in both the air pouch and the

intestine proinflammatory reactions through the activa-

tion of the cells forming the barriers (Fig. 2). In addition,

using FITC-labelled bacteria we showed that their strain

cell-specific interaction with the air pouch and intestine

GALT barrier associates only the strains exhibiting strong

immunostimulatory activity (Fig. 2). This interactivity is

the signal initiating the proinflammatory response by the

production of species-specific chemokines and cytokines

in the air pouch (Yam et al. 2008). Phagocytic activity

may also be a common function induced by Lactobacillus

in both intestine and air pouch. Our data have demon-

strated increased ex vivo phagocytic activity of the air

pouch PMN cells (Fig. 1) as well as increased in vivo

internalization of Lactobacillus antigens by lymphatic cells

localized in areas of Peyer’s patches harbouring dendritic

cells and macrophages (Fig. 2). Lactococcus lactis adminis-

tration in the air pouch increased PMN recruitment, while

Large intestine

Small intestine160

140

120

100

80

60N

umbe

r of

pos

itive

cel

ls/1

0 fie

lds

40

20

0 INF

-g

TN

F-a

IL-6

IL-10

IL-12

INF

-g

TN

F-a

IL-6

IL-10

IL-12

*

**

*

**#

**

*

*

*

*

*

*

*

*

(a)

(b) (c) (d)

Figure 4 Effect on the cytokine profile of the

small and large intestines of mice fed with

the assayed lactobacilli strains. (a) Determina-

tion of the number of cells producing

cytokines in the small and large intestines of

mice by immunofluorescence. The histogram

is representative of two similar experiments

and each bar represents the means ± SEM

(n = 5). *Statistically significant differences in

comparison with the control group, P < 0Æ05.#Statistically significant differences in

comparison with the probiotic NCFB 1748-

treated group, P < 0Æ05. (b–d) Immuno-

fluorescence microphotographs showing IFN-c

positive cells in the small intestine of (b) unfed

mice and in (c) the small or (d) the large

intestine of DC412-fed mice. ( ) Normal;

( ) NCFB 1748; ( ) DC412 and ( ) DC205.

A. Kourelis et al. Probiotic injection in mouse air pouch

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284 281

PMN recruitment in the udder because of L. lactis

administration successfully treated bovine mastitis (Crispie

et al. 2008), suggesting that DC412 and NCFB 1748

strains may also exhibit pathogenic bacterial clearance

activity. Another similarity between the air pouch and the

intestine barriers is the increase in TNF-a production in

both the air pouch and the small intestine in response to

DC412 and NCFB 1748 administration, suggesting that

this proinflammatory cytokine may be considered among

the major initial signals required for the activation of

innate immune response by these strains. The increase in

TNF-a level may be required to initiate the interaction

between the intima and subintima cells of the air pouch

as this mechanism is also suggested for intestine epithelial

cells (Galdeano et al. 2007). The precise mechanism by

which DC412 and NCFB 1748 administration in the air

pouch resulted in increased PMN recruitment, phagocytic

activity and cytokine production remains unclear in this

study. Possible mechanism could include the activation of

chemokines such as MIP-2 (Walley et al. 1997; Yam et al.

2008), TLRs (Doyle et al. 2004) and ⁄ or adjuvant activity

(Vitini et al. 2000).

Based on the above, we propose the eligibility of the

air pouch model to discriminate presumptive probiotic

Lactobacillus strains exhibiting immunostimulatory acti-

vity. The efficiency of the air pouch model to discrimi-

nate micro-organisms possessing probiotic properties

with respect to their immunomodulatory activity has

been also successfully shown for other Lactobacillus spe-

cies as well as for yeast strains in the unpublished studies

of our group (C. Kotzamanidis 2009, A. Kourelis 2009).

Our studies also showed differences concerning the

inflammatory response in the air pouch versus the intes-

tine. On the basis of PMN recruitment, air pouch

administration of Lactobacillus strains activates pro-

inflammatory reaction of higher potency than the

respective response observed in the intestine after oral

administration, suggesting enhancing capacity of the air

pouch on the innate immune responses. This may be

because of higher number of resident immune cells as

well as bacterial antigen availability in the air pouch

than in the intestine. However, in the intestine, the

Lactobacillus internalization is not followed by activation

of a general inflammatory response because oral DC412

Large intestine

Small intestine

140

120

100

80

60

Num

ber

of p

ositi

ve c

ells

/10

field

s

40

20

0 TLR

2

TLR

4

TLR

6

TLR

9

TLR

2

TLR

4

TLR

6

TLR

9

*

**

**

*

*

**

**

*#

*

*

(a)

(b) (c)

Figure 5 Effect of NCFB 1748, DC412 or

DC205 strains oral administration on the

number of Toll-like receptor (TLR) positive

cells in small and large intestines. (a) Determi-

nation of the number of TLR positive cells in

the small and large intestines of mice by

immunofluorescence. The histogram is

representative of two similar experiments and

each bar represents the means ± SEM (n = 5).

*Statistically significant differences in

comparison with the control group, P < 0Æ05.#Statistically significant differences in

comparison with the probiotic NCFB 1748-

treated group, P < 0Æ05. (b–c) Immuno-

fluorescence microphotographs showing TLR9

positive cells in the small intestine lamina

propria crypts of (b) unfed mice or (c) NCFB

1748 fed mice, arrows show the TLR9 positive

Paneth cells on the base of crypts. ( )

Normal; ( ) NCFB 1748; ( ) DC412 and

( ) DC205.

Probiotic injection in mouse air pouch A. Kourelis et al.

282 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 274–284

ª 2009 The Authors

and NCFB 1748 delivery does not affect the structure of

the villi and the massive influx of PMN (Fig. 3a–c) as

well as the number of cells producing the inflammatory

mediators COX-1 and COX-2 (Fig. 3d). Thus, the signi-

ficance of the route of the DC412 and NCFB 1748

delivery may reflect their potential immunoadjuvant or

immunoregulatory activity.

Oral administration of DC412 strain resulted in

increased immune responses concerning IgA (Fig. 3) and

cytokine production (Fig. 4) confirming that the data

obtained in the air pouch show immunoregulatory capa-

city of this strain. The DC205 strain that was a low

responder when administered in the air pouch also

responded poorly after its oral administration. Activation

of the innate immunity and IgA production are thought

to be the most important mechanisms involved in gut

mucosa immune stimulation by probiotics (Galdeano

et al. 2007). The involvement of TLRs in the mechanism

that activates the innate immune response in the gut

mucosa is well documented (Takeda et al. 2003). Our

findings demonstrated that DC412 induced the produc-

tion of IgA, TNF-a, IFN-c, IL-6 and IL-10 by small

intestine cells, and our studies indicate that this profile

could be mediated through TLR2 and TLR4 (Fig. 5). The

cytokine profile of IFN-c, TNF-a, IL-6, IL-10 and IL-12B

that is induced in the mouse intestine after oral adminis-

tration of the probiotic NCFB 1748 strain could be medi-

ated through TLR2 and TLR9. CpG sequences activate a

TLR9-mediated signalling cascade with subsequent release

of pro-inflammatory cytokines such as TNF-a, IL-6 and

IL-12 (Krieg 2002). Thus, the increased IL-12B observed

in NCFB 1748-treated mice could be because of the acti-

vation through intestinal epithelial cell surface TLR9

(Ewaschuk et al. 2007) or through macrophage ⁄ dendritic

cell endoplasmic TLR9 (Latz et al. 2004). These data are

in agreement with previous data concerning the cytokine

profile activated by LAFTI L10 (Lact. acidophilus) and

LAFTI L26 (Lact. paracasei) strains in mouse small

intestine (Paturi et al. 2007). Thus, the cytokine and TLR

pattern profiles observed in DC412 and NCFB 1748

strains are suggestive of potential differential activity in

which both strains promote balanced Th1 and Th2

immune responses. In summary, the DC412 as well as the

NCFB 1748 strains exhibit similar in vitro probiotic prop-

erties and immunostimulatory activity in the air pouch

and the gut mucosa with the exception of TLR9 expres-

sion, suggesting potential differential activity. Thus, the

DC412 strain may be considered as presumptive probiotic

candidate that differs from NCFB 1748.

In conclusion, the Lact. paracasei subsp. paracasei

DC412 strain exhibited regulatory activity of early

immune responses in both air pouch and intestine, which

is characterized by stimulation of PMN chemotaxis, phago-

cytic activity, TLR2 ⁄ TLR4 signalling and secretion of a cer-

tain cytokine profile. The dorsal air pouch system provides

the environment that permits the examination and predic-

tion of innate immune reactions in the gut and thus may

be used as a model to rapidly and efficiently discriminate

potential probiotic Lactobacillus strains.

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