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Effect of orally administered probiotic E. coli strain Nissle 1917 on intestinal mucosal immune cells of healthy young pigs
Veterinary Immunology and Immunopathology 111 (2006) 239–250 www.elsevier.com/locate/vetimm
Effect of orally administered probiotic E. coli strain Nissle 1917 on intestinal mucosal immune cells of healthy young pigs Swantje C. Duncker a,1, Axel Lorentz b, Bernd Schroeder a, Gerhard Breves a, Stephan C. Bischoff b,* b
a Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hannover, Germany Department of Nutritional Medicine and Prevention, University of Hohenheim, Fruwirthstr. 12, 70593 Stuttgart, Germany
Received 8 August 2005; received in revised form 8 December 2005; accepted 16 January 2006
Abstract Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients; however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered orally to young pigs at two concentrations (109 and 1011 CFU/d for 21 days) on the gut-associated lymphatic tissue. This probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin, prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (1011 CFU/d) showing an increase in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple modulation of immune cell distribution. # 2006 Elsevier B.V. All rights reserved. Keywords: Probiotics; Intestine; Porcine; GALT; Lymphocytes; E. coli Nissle 1917; CD8
Abbreviations: AMP, anti-microbial peptide; CD, cluster of differentiation; EcN, Escherichia coli Nissle 1917; GALT, gut associated lymphoid tissue; IEL, intraepithelial lymphocytes; LP, lamina propria; UC, ulcerative colitis * Corresponding author. Tel.: +49 711 459 4101; fax: +49 711 459 4343. E-mail addresses: [email protected] (S.C. Duncker), [email protected] (S.C. Bischoff). 1 Present address: Brain-Body Institute (BBI), 50 Charlton Ave. E, Hamilton, Ont., Canada L8N 4A6. Tel.: +1 905 522 1155x2277/5284. 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.01.017
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1. Introduction Particular probiotic strains have been successfully used for prophylaxis of intestinal infection in livestock animals (Vanbelle et al., 1990; Alexopoulos et al., 2004). In humans, they are known to prevent atopic dermatitis (Lodinova-Zadnikova et al., 2003; Kallioma¨ki et al., 2003), gastrointestinal infectious and antibiotic associated diarrhoea (McFarland et al., 1995; D’Souza et al., 2002), as well as inflammatory bowel disease such as ulcerative colitis (Kruis et al., 1997, 2004; Rembacken et al., 1999). Furthermore, some probiotics such as Lactococcus lactis have been used for delivery of cytokines towards the intestinal mucosa (Steidler et al., 2003). Even though selective probiotics such as Lactobacillus GG (Kallioma¨ki et al., 2001, 2003), Saccharomyces boulardii (McFarland et al., 1995) and E. coli Nissle 1917 (Kruis et al., 1997, 2004; Rembacken et al., 1999) have been proven to be clinically effective, the modes of action by which they achieve their beneficial effects remained unclear. In vitro experiments suggested that both modulation of the gut flora and of the intestinal cytokine production subsequently leading to modulation of the adaptive immune response might be of relevance (Christensen et al., 2002; Vaarala, 2003). So far, only a few studies have investigated the influence of probiotics on the number and distribution of intestinal immune cells in vivo (Perdigon et al., 1995; Pestka et al., 2001). The probiotic E. coli strain Nissle 1917 (EcN) used in this study is of the serotype O6:K5:H1 and was isolated in 1916 by the German physician Alfred Nissle (Loew, 2000). Since then this bacterial strain has been used as a probiotic drug and is considered to be safe (Blum et al., 1995; Grozdanov et al., 2002, 2004; Westendorf et al., 2005). Oral administration of up to 7.2 109 CFU of EcN per animal did not cause colitis or urogenital infection in neonatal gnotobiotic piglets (Gunzer et al., 2002). EcN contains different so-called ‘‘fitness factors’’ such as siderophores and microcins (Patzer et al., 2003; Grozdanov et al., 2004). Moreover, the bacterium shows a distinct LPS-structure supposed to exert particular immune modulating properties (Grozdanov et al., 2002). EcN is the active component of Mutaflor1, a drug showing equivalent effectiveness as 50 -amino-salicylates in maintaining remission in
ulcerative colitis (UC) in humans (Kruis et al., 1997, 2004; Rembacken et al., 1999). The mechanisms underlying the beneficial effects of EcN are unclear as yet, immunomodulating effects have been discussed (Hockertz, 1997; Schultz et al., 2004; Sturm et al., 2005). Here, we aimed to further elucidate the mechanisms of action in an animal system thought to have a rather similar intestinal anatomy and structure of the mucosal immune system compared to man. This seems to be of relevance, since beneficial clinical effects of EcN were only shown in humans and calves (von Buenau et al., 2005) so far. Therefore, we chose pigs for our ex vivo studies and examined the effects of EcN on immune cell distribution and cytokine expression as well as AMP (antimicrobial peptides) expression in the intestinal mucosa.
2. Material and methods 2.1. Animals Fifteen young adult pigs with a mean weight of 16 2.7 kg were used for experiments. The pigs originated from different litters of a conventional crossbred program of ‘‘German Landrace’’ and ‘‘Pietrain’’ carried out by the university research farm. All animals were on a regular deworming schedule with 0.3 mg/kg ivermectin two times a year. Upon their arrival the pigs were housed on straw. They were maintained on a regular mixed diet for growing pigs. Following a 2 weeks adaptation period the animals were divided into three groups of five pigs each. The first group served as control. The second group received 109 CFU EcN/d and the third group 1011 CFU EcN/d. EcN was given as a suspension in dilution medium (2.5 g NaCl, 2.5 g KCl, 0.2 g MgSO4, 0.2 g CaCl2, 0.2 g MgCl26H2O, 20 ml NaOH (32%) add distilled H2O to a total of 1000 ml) orally for 21 days. The control group received the dilution medium without EcN. The animals were killed by exsanguination following mechanical stunning. The abdominal contents were removed immediately after death. The protocol of animal treatment was approved and the procedure was supervised by an animal protection officer of the University.
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2.2. Fecal samples
2.4. Histology
To ensure proper administration of EcN, fecal samples were taken from every animal at days 7 and 14 of the experiment and after slaughter. They were analyzed for EcN content by multiplex PCR using strain-specific DNA primer sequences as described elsewhere (Blum-Oehler et al., 2003). All EcN-treated animals were tested positive for EcN at days 7 and 14 whereas control animals always yielded negative EcN RT-PCR results (data not shown).
Paraformaldehyde-fixed paraffin slides were deparaffinized, hydrated and stained using Meyer’s hemalum solution (Merck, Darmstadt, Germany) and aqueous eosin Y Solution (Sigma–Aldrich, Steinheim, Germany) according to standard protocols (Romeis, 1989). Carnoy’s solution fixed paraffin sections were stained with toluidine blue O (Sigma– Aldrich) for an hour and counterstained with Meyer’s haemalum. Following dehydration with increasing concentrations of alcohol and finally xylene, the slides were mounted in Entellan1 (Merck).
2.3. Tissue samples 2.5. Monoclonal antibodies Tissue samples were taken immediately after slaughter from the duodenum (10 cm aboral from the pylorus), jejunum (4.50 m aboral from the pylorus), ileum (5 cm oral from the ileo–colic orifice), ascending (10 cm aboral from the caeco– colic junction) and descending (60 cm aboral from the end of the ascending colon) colon. Mucosa preparations and small tissue specimen (0.5 cm) were immediately snap frozen in liquid nitrogen for RNA isolation and cryostat sectioning, respectively. An additional tissue specimen was collected at each location (0.5 cm) and fixed either in 4% paraformaldehyde at pH 7.2 for 18–24 h or in Carnoy’s solution (60% ethanol absolute, 30% chloroform, 10% glacial acetic acid) for 2 h. The samples were embedded in paraffin wax and sectioned at 2 mm, placed on SuperfrostTM plus slides (Menzel, Braunschweig, Germany) and dried overnight at 37 8C.
The antibodies used in this study are listed in Table 1. Biotinylated secondary Ab (sAb) were obtained from Southern Biotechnology, Birmingham, USA. 2.6. Immunohistochemistry Different immune cell populations were identified by immunohistochemistry using the enzyme-linked immunoperoxidase technique. Incubation steps were carried out in a humid chamber at room temperature if not indicated otherwise. After each step slides were washed for 5 min in TBS (pH 7.4) supplemented with 0.035% Tween120 (Sigma–Aldrich). CD4+ and CD8+ subpopulation analysis was performed using cryostat sections of 5 mm thickness placed on Superfrost1 plus glass slides (Menzel-Glaeser, Braunschweig, Germany). Sections were air-dried
Table 1 Antibody dilutions Primary Ab CD4 CD8 CD25 IgA Mouse IgG1 Mouse IgG2a Mouse IgG2b a b
Clone 74-12-4 MIL-12 231.3B2 K61 1B4 – – –
Company VMRD, USA Serotec, UK Serotec, UK Serotec, UK Dianova, Germany Dianova, Germany Dianova, Germany
Dilutions were prepared with Monet Blue (Biocare, Germany). Dilutions were prepared with VanGogh Yellow (Biocare, Germany).
Dilution a
1/50 1/100 b 1/20a 1/10a 1/20a 1/100 c 1/50b
Secondary Ab Goat–anti-mouse IgG2b Goat–anti-mouse IgG2a Goat–anti-mouse IgG1 Goat–anti-mouse IgG1 Goat–anti- mouse IgG1 Goat–anti-mouse IgG2a Goat–anti-mouse IgG2b
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for 30 min and fixed in a mix of equal amounts of acetone and methanol at 4 8C. Tissues were blocked with 5% normal swine serum diluted in Tris-buffered saline (TBS) for 30 min to reduce unspecific Abbinding, and then incubated overnight with the primary Ab at 4 8C. The following day slides were blocked again with 5% normal swine serum diluted in TBS, and then incubated with a biotinylated secondary Ab (1/50 dilution) for 30 min. Streptavidin–horse radish peroxidase (Biocare, Hamburg, Germany) and 3-amino-9-ethyl carbazol (AEC) were used to visualize specific binding of the Ab (Zymed, San Francisco, CA). Slides were counterstained with Meyer’s hemalum solution for improved cell differentiation and finally mounted in Faramount1 Aqueous Mounting Medium (Dako, Hamburg, Germany). Each staining series was accompanied by one slide treated with an isotype control replacing the primary Ab. Because of the lack of endogenous peroxidase activity in the cryostat sections, blocking with hydrogen peroxide was not necessary using this staining protocol. For the detection of CD25 and IgA paraffin embedded specimen were used. Slides were deparaffinized and hydrated. To ensure antigen de-masking the samples were treated with Dako1 Target Retrival Solution (pH 6) (Dako) in a water bath at 95 8C. The subsequent immunohistochemistry was performed as described above; however, an additional incubation in methanol containing 1.2% hydrogen peroxide was added following the incubation with the sAb to ensure quenching of endogenous peroxidase. 2.7. Cell counts Cells counts within the intestinal mucosa were performed at 1000-fold magnification using a light microscope Labophot-2 (Nikon, Tokyo, Japan). Cells were counted separately in the epithelium and the lamina propria (LP). Data were expressed in percent of positive cells per total number of epithelial cells or total number of cells in the LP, respectively. Cells were assigned to the intraepithelial compartment only if they were located with the epithelial cell band or at least half of their plasma membrane was in contact with the basement membrane of the epithelium. For CD8+ cells, we differentiated between luminal and basal section of the mucosa. Therefore, the luminal
compartment of the mucosa was considered to be the villi in the small intestine and the most luminal microscopic field of view (1000 times magnification) in the large intestine. The basal compartment of the mucosa contained the crypts in the small intestine and the most basal microscopic field of view in the large intestine. The assignment made in the large intestine ensured that cells were not accidentally counted for both compartments. CD4+ cells could be evaluated on a semi-quantitative basis only by assignment to four groups defined as follows: (1) <25% of LP cells CD4+; (2) 25–49% of LP cells CD4+; (3) 50–75% of LP cells CD4+; (4) >75% of LP cells CD4+. 2.8. RNA isolation and RT-PCR Total RNA was prepared from washed and stripped mucosa samples (0.9–0.8 g) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s manual. The amount of total RNA was quantified by spectrometry. For RT-PCR 200 ng of total RNA was used and processed as described previously (Gebhardt et al., 2002). Briefly, RNA was treated with 10 U RNase-free DNAse (Promega, Madison, WI) to remove genomic DNA. After denaturation at 70 8C cDNA was synthesized at 49 8C adding SuperscriptTM plus reverse transcriptase (Invitrogen, Karlsruhe, Germany) and oligo-dT primers (Pharmacia, Uppsala, Sweden). 1/10 of the total cDNA was used for one PCR reaction. PCR was performed with 30 s at 94 8C, 45 s at 55–58 8C, 35 s at 72 8C. Numbers of cycles and annealing temperatures differed for each pair of primers: IFN-gamma (34 cycles, annealing 58 8C), TNF-alpha (35 cycles, annealing 58 8C), TGF-beta (32 cycles, annealing 58 8C), IL-10 (40 cycles, annealing 58 8C) preprodefensin-beta 1 (39 cycles, annealing 58 8C), protegrins and PR-39 (42 cycles, annealing 55 8C), NKlysin (36 cycles, annealing 55 8C) and GAPDH (28 cycles, annealing 56 8C). To ensure that a saturation of primers were not misinterpreted as an increase of mRNA, as many cycles of PCR were used as were necessary to ensure the amplification reaction was still in its exponential phase. The number of cycles for every primer pair was determined by preliminary experiments with samples from at least three different untreated pigs and for every intestinal location tested. The cycle number was than used in treated and
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Table 2 PCR-primer
IFN-gamma TNF-alpha TGF-beta IL-10 PR-39 NK-lysin Protegrines Prepro-defensine-beta GAPDH
Sense-primer
Antisense-primer
50 -GCAAGTACCTCAGATGTACC-30 50 -ACTGCACTTCGAGGTTATCG-30 50 -CAGAGAGGCTATAGAGGGTT-30 50 -TTGCCAAGCCTTGTCAGAGA-30 50 -ACCCATCCATTCACTCAC-30 50 -GAGCAGTTCTGCCAGAACCT-30 50 -TGGATCAGATCAAGGACC-30 50 -CCTCCTTGTATTCCTCCTCA-30 50 -ACCACAGTCCATGCCATCAC-30
50 -TGGCCTTGGAACATAGTCTG-30 50 -AGAGGACCTGGGAGTAGATG-30 50 -TGTCTAGGCTCCAGATGTAG-30 50 -TCACCCATGGCTTTGTAGAC-30 50 -AGCCACAACAATAAGATCC-30 50 -GCAGGAGTTAGGTGAGAGAA-30 50 -ACACAGACGCAGAACCTAC-30 50 -TTGCAGCATTTGACTTGGGG-30 50 -TCCACCACCCTGTTGCTGTA-30
untreated animals to allow comparative semi-quantitative detection of mRNA expression. PCR was performed with 2.5 U Taq DNA polymerase (Invitrogen) and 20 pmol of specific sense and antisense primers shown in Table 2. The PCR products contained cDNA fragments of 359 bp (IFN-gamma), 266 bp (TNF-alpha), 371 bp (TGF-beta), 243 bp (IL-10), 168 bp (prepro-defensin-beta 1), 262 bp (PR-39), 100 bp (protegrins), 479 bp (NK-lysin) and 452 bp (GAPDH), respectively. Eleven microliters of the PCR products were visualized on 1% agarose gel containing ethidium bromide (500 ng/ml) and photographed.
significantly different when comparing small and large intestinal sites, their density within the LP ranging from 0.4% in the jejunum to 3.7% in the duodenum and colon (Fig. 1B). Mast cells were equally distributed throughout the intestinal LP, and accounted for about 3% of LP cells at all
2.9. Statistics Data were expressed as mean (S.D.) if not indicated otherwise. Differences in cell counts were accessed using the Student’s t-test and differences between experimental groups by one-way ANOVA.
3. Results 3.1. Number and distribution of granulocytes and mast cells In untreated animals, granulocytes and mast cells were detected in the lamina propria of the mucosa, but not in the epithelium. Eosinophils were concentrated within the basal part of the LP. They were much more frequent in the small compared to the large intestine, with highest numbers in the mucosa of the ileum (11 2.2% of total LP cells). In the colon almost no eosinophils were visible (0.6 0.3% of total LP cells) (Fig. 1A). Neutrophil numbers were not
Fig. 1. Eosinophil (A), neutrophil (B) and mast cell (C) density in the intestinal lamina propria mucosae (LP) shown as per cent of all lamina propria cells. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). Bars show the mean of five pigs per group with error bars indicating the standard deviation.
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investigated sites of the gut (Fig. 1C). No basophils could be detected in the intestinal mucosa of the pigs. The numbers of the three cell types examined here did not change following treatment of the animals with either low or high dose of EcN, as shown in Fig. 1. 3.2. Number and distribution of T lymphocyte subsets and plasma cells IgA producing plasma cells, CD4+ cells, CD25+ cells and CD8+ cells were analyzed by immunohistochemistry. IgA plasma cells were predominantly found in the LP of the crypts. They were virtually absent in the epithelium and in submucosal layers. Number of IgA plasma cells were highest in the duodenum with a density of 21.1% (3.1% of LP cells). Plasma cell counts were significantly lower at
Fig. 2. Density of IgA+ plasma cell (A), CD4+ cells (B) and CD25+ cells (C) in the intestinal lamina propria mucosae (LP) shown as per cent of all lamina propria cells. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). Bars show the mean of five pigs per group with error bars indicating the standard deviation.
the more caudal sites of the GI tract (jejunum, ileum, colon), where only 3% of LP cells were IgA plasma cells. The density of IgA plasma cells was not changed by treatment of the animals with EcN (Fig. 2A). Cells expressing the CD4 co-receptor were frequently found in the LP but were absent in the epithelium. Within the LP 50–75% of cells were CD4 positive. Therefore, we only performed a semiquantitative analysis. CD4+ cells were evenly distributed throughout cross-sections of the mucosa in the ileum and colon; however, the mucosa of the duodenum and jejunum contained higher numbers of CD4+ cells in the LP of the villi compared to the crypts (Fig. 2B). CD25+ cells were localized in small amounts (2.4–3.7% of total LP cells) in the intestinal mucosa. They were evenly distributed in the LP (Fig. 2C) and only sporadically detected in the epithelium. Again, neither CD4+ cell density nor CD25+ cell density changed following EcN treatment (Fig. 2B and C). CD8+ cells were the only ones found to be consistently present within the epithelium and the LP of the intestinal mucosa. The density of CD8+ cell was higher in the luminal compared to the basal
Fig. 3. Density of CD8+ cells in the luminal compartment of the mucosa (A) and the basal compartment of the mucosa (B), shown in percent of all epithelial cells, LP cells, respectively. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). First bar of every colour pattern indicating cells in the epithelium and the second bar cells in the LP. Bars show the mean of five pigs per group with error bars indicating the standard deviation. *p < 0.05 compared to same intestinal segment in the control group.
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compartment of the mucosa. Highest counts were found in the luminal epithelium of the duodenum. Along the intestinal mucosa the CD8+ cell counts in the LP were reverse to those in the epithelium of the same compartment. For example in the luminal compartment of the mucosa the epithelium contained much higher numbers of CD8+ cells than the corresponding LP (Fig. 3A). In the basal compartment of the mucosa the opposite was true (Fig. 3B). Animals receiving high amounts of EcN
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(1011 CFU/d) showed significantly higher CD8+ cells counts in the luminal LP of the Ileum, in the epithelium and the lamina propria of the ascending colon compared to placebo-fed pigs (Figs. 3 and 4). In low dose fed animals CD8+ cells where increased in the basal epithelium of the jejunum and in the luminal LP of the ascending colon (Fig. 3). No significant changes in CD8+ cell counts were observed in the mucosa of other intestinal segments on EcN treatment.
Fig. 4. Pictures showing cells bearing the CD8 antigen (red) in the mucosa of the ascending colon. The animal treated with 1011 CFU/d (B) of EcN shows an increase in the number of CD8 positive cells compared to the untreated animal (A). Slides were stained by immunohistochemistry using the MIL-12 Ab (Serotec, UK) or a mouse IgG2a (Dianova, Germany) isotype as negative control (C). Pictures from one representative animal per group are shown. Metering bar = 25 mm.
Fig. 5. mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (prepro-defensin-beta 1, NKlysin) in the ileum and the ascending colon compared to housekeeping gene (GAPDH). Pictures show two representative animals of the control group and the group treated with 1011 CFU/d of E. coli Nissle 1917 (EcN). Numbers indicate the PCR cycles accomplished.
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3.3. mRNA expression of different cytokines and AMP Total RNA from intestinal mucosa of duodenum, ileum and ascending colon in control animals and animals fed 1011 CFU EcN/d was investigated. mRNA expression of different cytokines and antimicrobial peptides was evaluated semi-quantitatively by comparison to GAPDH as housekeeping gene to check for possible changes in immune cell subpopulations. mRNA expression of IFN-gamma, TNFalpha, TGF-beta and IL-10 as well as the mRNA expression of the AMP: prepro-defensine-beta 1 and NK-lysin was detected in the duodenum (data not shown), ileum and ascending colon (Fig. 5). In all samples PR-39 was only partly seen in the intestinal mucosa and mRNA for protegrins was absent in all animals and intestinal segments (data not shown). Transmural duodenal tissue samples of 3-week-old piglets were included as positive controls. Neither in the investigated cytokines nor in the AMP changes were detected after feeding high amounts of EcN (1011 CFU/d).
4. Discussion During the last decades pigs health became a huge economic factor in livestock farming because of intensive animal husbandry and increasing production rates. Simultaneously, the opportunities of prophylactic treatment against the major porcine pathogens were limited due to governmental restriction (prohibition of antibiotics in animal feed, etc.). Therefore, alternatives are needed. Probiotics showed the potential to fulfil such requirements, even though the mechanisms of their beneficial effects are mostly unknown. This study was performed to investigate the effects of EcN, a probiotic E coli strain successfully used in human and veterinary medicine, on the intestinal immune system of healthy young pigs. The aim was to further understand the modes of action of this probiotic strain, which obviously has beneficial properties both in man (Kruis et al., 2004) and in calves (von Buenau et al., 2005). We selected the pig as a model not only because this species might as well benefit of probiotic treatment (Alexopoulos et al., 2004), but also because the pig intestinal immune
system is considered to be much closer to the human one than the rodent immune system (Miller and Ullrey, 1987; Boes and Helwigh, 2000). Studies have been performed in pigs on the distribution of particular immune cells in the blood and in different tissues (Bianchi et al., 1992; Yang and Parkhouse, 1996; Boeker et al., 1999; Saalmuller et al., 2002; Sinkora et al., 2005) including studies on T cells in the intestine (Vega-Lopez et al., 1993; Pabst and Rothko¨tter, 1999). Nevertheless, it is surprising to note that no published data are available on the distribution of cells of the innate immune system within the intestine of the pig. Therefore, we were interested not only in studying the influence of EcN on intestinal immune cells, but also in quantifying the number and distribution of granulocytes, mast cells and lymphocyte subsets in the gut mucosa. Eosinophils were mainly found in the LP of the crypts in the small intestine and the basal part of the crypts in the large intestine, as described previously (Bianchi et al., 1992). Eosinophils could be found at all sites of the small and large intestine, the highest numbers being seen in the ileum. Interestingly, we found for the first time that the density of eosinophils in the colon LP was lower than 1% and thus much lower than that described in humans (Bischoff et al., 1996). Because none of the animals showed signs of parasite infection and all were dewormed on a routine schedule, an increase of eosinophils in the small intestine due to parasitosis can be largely ruled out. There is evidence from the literature that gastrointestinal eosinophils not only act as inflammatory effector cells of allergy and parasitic infection, but are also capable of functioning as immunomodulating cells (Kato et al., 1998). Kato et al. showed a spontaneous minimal degranulation of intestinal eosinophils in the healthy gut that does not occur in other normal tissues. These data suggest that eosinophils may also act as immune-modulating cells in the small intestine of pigs. Compared to eosinophils the amount of neutrophils in the intestinal mucosa of healthy pigs was much lower. Neutrophil counts were highest in the duodenum and descending colon. The amount of neutrophils was higher than the numbers regarded to be normal in humans (0.4%) (Ogra et al., 1999). In the duodenum the reason might be that pigs are more prone to contact with microorganisms because
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of their usual environment. Therefore, more neutrophils may ensure a fast reaction to potential pathogens. The relatively high amount of neutrophils in the colon, compared to humans, is likely to be a result of higher physiologic amounts of bacteria in this part of the pig intestine compared to the human colon. In contrast to neutrophils, the counts of mast cells were in the expected ranges, since our data were similar to those reported by Xu et al. (1993) who did similar studies in pigs. Mast cells, unlike granulocytes, were evenly distributed throughout the intestinal segments. Treatment with EcN had no effect on the number and distribution of granulocytes and mast cells in healthy pigs. Moreover, the absence of major changes in tested cytokine and AMP mRNA expression makes an influence of EcN on the cellular metabolism unlikely too. Even though, a definite conclusion on cytokine-mRNA concentration would require a quantitative method for mRNA detection like real time PCR, because the semi-quantitative method used can only detect major changes. However, the unaltered number of cells in healthy EcN-treated animals does not rule out a positive effect on disease-induced cell recruitment (Sturm et al., 2005; Kamada et al., 2005). From experiments with other probiotics like lactobacilli it is known, that they are able to decrease infection-induced, transmucosal migration of neutrophils (Michail and Abernathy, 2002). Keeping in mind EcN being a probiotic, the fact that it does not recruit neutrophils in healthy animals rather underlines its apathogenicity than its ineffectiveness. Even though in the present study no changes were detected with respect to the number and distribution of intestinal eosinophils, it still needs to be clarified if the administration of EcN leads to alterations in mediator composition and release in this cells. In accordance with findings of other authors (Rothko¨tter et al., 1991; Bianchi et al., 1992) IgAproducing plasma cells were mainly found in the basal part of the mucosa with highest amounts in the crypts of the duodenum. No changes were detected following EcN treatment. An increase in the number of intestinal IgA producing plasma cells, does not seem to be a mechanism of action of EcN in conventionally reared, healthy pigs. Nevertheless, unchanged IgA plasma cell counts do not rule out an influence of EcN on the luminal secretory IgA concentration as reported for
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other probiotics like Saccharomyces boulardii (Buts et al., 1990; Rodrigues et al., 2000). Heterogeneous results were obtained with regard to lymphocyte subsets. We found no changes in CD4+ and CD25+ cells, but an increase of CD8+ cells in the ascending colon induced by EcN. In contrast to the results of others (Vega-Lopez et al., 1993), we found in our experiments polarization of mucosal CD4+ cells towards the LP of the villi only in the duodenum and jejunum. In the ileum and colon, the CD4+ lymphocytes were equally distributed throughout the mucosa. An explanation of this difference might be the lower age of the animals used in the study by Vega-Lopez et al. Even though no alterations in the CD4+ cell counts were evident between treatment and control groups, an influence EcN on the balance of CD4+ subpopulations cannot be ruled out. Different from CD4+ cells significantly more CD8+ cells were counted in the luminal epithelium than in the lamina propria of the intestine, as reported earlier by other groups (Vega-Lopez et al., 1993, 2001; Pabst and Rothko¨tter, 1999). This holds true for all intestinal segments investigated. We extended these findings by distinguishing between the epithelium and the LP in the luminal and the basal part of the mucosa, respectively. The highest number of CD8+ cells (one-third of total cell count) was found in the luminal epithelium of the duodenum. Different from the findings of Vega-Lopez et al. we could not confirm that the CD8+ cells of the LP were localized near the basement membrane of the epithelium, possibly because of different methodological approaches. We report here for the first time that EcN treatment increased the amount of CD8+ cells in the ascending colon in healthy young pigs. Feeding 1011 CFU EcN/d for 21 days resulted in an increase of CD8+ cells in the ascending colon, both in the luminal as well as in the basal compartment of the mucosa. In the absence of mucosal inflammation (no increase in neutrophils, no differences of proinflammatory cytokine-mRNA expression in comparison between treated and untreated groups, no activation of lymphocytes) the high numbers of CD8+ cells cannot be interpreted as an inflammatory response. From our experiments it cannot be concluded whether the increased number of CD8+ cells detected in the epithelium of the ascending colon is due to proliferation of CD8+ cells or to recruitment of cells from other sites like the lamina
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propria or the mesenteric lymph nodes. However, it has been shown in mice that intestinal IEL are able to proliferate in the epithelium (Lin et al., 1994). The accumulation of lymphocytes originating from the peripheral blood as an explanation of the increase of CD8+ cells due to EcN treatment is unlikely, since Sturm et al. (2005) showed a down-regulation of proliferation in peripheral blood T cells by EcN in vitro, whereas the proliferation of LP T cells was unchanged. The cell population expressing CD8 coreceptor in the intestine consists of different cell types including cytotoxic T cells, gamma/delta T cells and NK cells. In this study we did not differentiate between subtypes of CD8+ cells as CD8high and CD8low. Therefore, no conclusion can be drawn on changes within a special subtype of CD8+ cells; however, the beneficial effects of probiotics such as EcN could be related to known effects of CD8+ subpopulations, such as defence against pathogens by cytotoxic CD8+ cells, or maintenance and enhancement of the epithelial integrity by gamma/delta CD8+ cells (Chen et al., 2002). Other than in humans and mice intraepithelial CD8+ gamma/delta T cells have not yet been identified in the intestine of pigs, but that does not rule out the possibility that equivalent subtypes of CD8+ cells exist also in pigs within the epithelium. Especially, because there is increasing evidence that pigs have a distinct repertoire of intestinal gamma/delta TCR (Holtmeier et al., 2002) and T cells expressing the delta chain of the TCR have been shown in the colonic epithelium (Hontecillas et al., 2005). Future experiments are needed to further characterize the phenotype of CD8+ cells in the pig, and to establish their biological relevance in disease and disease prevention. Even though the porcine intestinal immune system might be an excellent model in terms of transfer of results to the situation in humans, there are of course some limitations. The lack of EcN effects on the density of innate immune cells such as granulocytes or mast cells shown in this study, as well as on the density of CD4+ cells (mostly T helper lymphocytes), CD25+ (activated T cells) and IgA cells might be the result of the limited number of animals examined here. Unfortunately, the repertoire of antibodies directed against porcine cell surface antigens is limited compared to that available for rodents. A future improvement in availability of
directly fluorescent-labeled antibodies to perform fluorescent immunohistochemistry and an improvement in isolation techniques for intestinal immune cells will be important to defining specialized subsets of lymphocytes and innate immune cells in situ. This will help to further investigate an influence of EcN on intestinal immune cells in the pig. Furthermore, using EcN in an infection model might be useful to reveal changes in the reaction and migration of intestinal immune cell population that cannot be detected in healthy animals.
5. Conclusions We showed in this study as described earlier in humans (Kruis et al., 2004) and mice (Waidmann et al., 2003; Schultz et al., 2004), that EcN did not induce any signs of inflammation and did not exhibit any signs of pathogenicity, even when fed to young pigs at high doses. The EcN-induced enhancement of the number of CD8+ cells in the ascending colon might be linked to the beneficial role of maintaining porcine intestinal health and preventing disease. Intestinal epithelial cells have been shown in vitro to produce keratinocyte growth factor (KGF) (Chen et al., 2002) and might thereby increase the epithelial integrity. Mice lacking gamma/delta TCR IEL have impaired epithelial development in the intestine (Boismenu and Havran, 1994). It has also been shown recently that IEL express junctional molecules like epithelial cells, whereas lymphocytes from other body sites (spleen, mesenteric lymph nodes, thymus, Payer’s patches) do not (Inagaki-Ohara et al., 2005). This opens up new possibilities for the involvement of IEL in the epithelial barrier and for the communication with epithelial cells. To further evaluate the beneficial effects of EcN in pigs and the underlying mechanisms, experiments with pig infection models are of special interest.
Acknowledgements We thank Gisela Weier and Gerhild Becker for excellent technical assistance and Yvonne Armbrecht and Michael Rohde for help with the animals as well as Anne Luschert for advice in immunohistochem-
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Effect of orally administered probiotic E. coli strain Nissle 1917 on intestinal mucosal immune cells of healthy young pigs Swantje C. Duncker a,1, Axel Lorentz b, Bernd Schroeder a, Gerhard Breves a, Stephan C. Bischoff b,* b
a Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hannover, Germany Department of Nutritional Medicine and Prevention, University of Hohenheim, Fruwirthstr. 12, 70593 Stuttgart, Germany
Received 8 August 2005; received in revised form 8 December 2005; accepted 16 January 2006
Abstract Several beneficial effects of probiotics have been described in studies using rodent disease models and in human patients; however, the underlying mechanisms remained mostly unclear. Only a few studies focused on the effects of probiotics on the intestinal mucosal immune system. Here, we studied the effect of the probiotic strain E. coli Nissle 1917 (EcN) administered orally to young pigs at two concentrations (109 and 1011 CFU/d for 21 days) on the gut-associated lymphatic tissue. This probiotic strain was shown recently to reduce recurrence of inflammation in ulcerative colitis patients. We quantified the number and distribution of intestinal immune cells (granulocytes, mast cells, CD4+, CD8+, CD25+, IgA+ lymphocytes) and the mucosal mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (PR-39, NK-lysin, prepro-defensin-beta 1, protegrins). The number and distribution of cells were highly different between small intestinal and colon segments in all groups, but were not influenced by EcN, except high dose EcN fed pigs (1011 CFU/d) showing an increase in mucosal CD8+ cells in the ascending colon. The mRNA analysis revealed no changes associated with EcN feeding. In conclusion, according to our analyses EcN has only minor effects on the distribution of mucosal immune cells in the gut of healthy individuals. The well-established preventive effects of EcN might therefore be relate to other mechanisms than simple modulation of immune cell distribution. # 2006 Elsevier B.V. All rights reserved. Keywords: Probiotics; Intestine; Porcine; GALT; Lymphocytes; E. coli Nissle 1917; CD8
Abbreviations: AMP, anti-microbial peptide; CD, cluster of differentiation; EcN, Escherichia coli Nissle 1917; GALT, gut associated lymphoid tissue; IEL, intraepithelial lymphocytes; LP, lamina propria; UC, ulcerative colitis * Corresponding author. Tel.: +49 711 459 4101; fax: +49 711 459 4343. E-mail addresses: [email protected] (S.C. Duncker), [email protected] (S.C. Bischoff). 1 Present address: Brain-Body Institute (BBI), 50 Charlton Ave. E, Hamilton, Ont., Canada L8N 4A6. Tel.: +1 905 522 1155x2277/5284. 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.01.017
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1. Introduction Particular probiotic strains have been successfully used for prophylaxis of intestinal infection in livestock animals (Vanbelle et al., 1990; Alexopoulos et al., 2004). In humans, they are known to prevent atopic dermatitis (Lodinova-Zadnikova et al., 2003; Kallioma¨ki et al., 2003), gastrointestinal infectious and antibiotic associated diarrhoea (McFarland et al., 1995; D’Souza et al., 2002), as well as inflammatory bowel disease such as ulcerative colitis (Kruis et al., 1997, 2004; Rembacken et al., 1999). Furthermore, some probiotics such as Lactococcus lactis have been used for delivery of cytokines towards the intestinal mucosa (Steidler et al., 2003). Even though selective probiotics such as Lactobacillus GG (Kallioma¨ki et al., 2001, 2003), Saccharomyces boulardii (McFarland et al., 1995) and E. coli Nissle 1917 (Kruis et al., 1997, 2004; Rembacken et al., 1999) have been proven to be clinically effective, the modes of action by which they achieve their beneficial effects remained unclear. In vitro experiments suggested that both modulation of the gut flora and of the intestinal cytokine production subsequently leading to modulation of the adaptive immune response might be of relevance (Christensen et al., 2002; Vaarala, 2003). So far, only a few studies have investigated the influence of probiotics on the number and distribution of intestinal immune cells in vivo (Perdigon et al., 1995; Pestka et al., 2001). The probiotic E. coli strain Nissle 1917 (EcN) used in this study is of the serotype O6:K5:H1 and was isolated in 1916 by the German physician Alfred Nissle (Loew, 2000). Since then this bacterial strain has been used as a probiotic drug and is considered to be safe (Blum et al., 1995; Grozdanov et al., 2002, 2004; Westendorf et al., 2005). Oral administration of up to 7.2 109 CFU of EcN per animal did not cause colitis or urogenital infection in neonatal gnotobiotic piglets (Gunzer et al., 2002). EcN contains different so-called ‘‘fitness factors’’ such as siderophores and microcins (Patzer et al., 2003; Grozdanov et al., 2004). Moreover, the bacterium shows a distinct LPS-structure supposed to exert particular immune modulating properties (Grozdanov et al., 2002). EcN is the active component of Mutaflor1, a drug showing equivalent effectiveness as 50 -amino-salicylates in maintaining remission in
ulcerative colitis (UC) in humans (Kruis et al., 1997, 2004; Rembacken et al., 1999). The mechanisms underlying the beneficial effects of EcN are unclear as yet, immunomodulating effects have been discussed (Hockertz, 1997; Schultz et al., 2004; Sturm et al., 2005). Here, we aimed to further elucidate the mechanisms of action in an animal system thought to have a rather similar intestinal anatomy and structure of the mucosal immune system compared to man. This seems to be of relevance, since beneficial clinical effects of EcN were only shown in humans and calves (von Buenau et al., 2005) so far. Therefore, we chose pigs for our ex vivo studies and examined the effects of EcN on immune cell distribution and cytokine expression as well as AMP (antimicrobial peptides) expression in the intestinal mucosa.
2. Material and methods 2.1. Animals Fifteen young adult pigs with a mean weight of 16 2.7 kg were used for experiments. The pigs originated from different litters of a conventional crossbred program of ‘‘German Landrace’’ and ‘‘Pietrain’’ carried out by the university research farm. All animals were on a regular deworming schedule with 0.3 mg/kg ivermectin two times a year. Upon their arrival the pigs were housed on straw. They were maintained on a regular mixed diet for growing pigs. Following a 2 weeks adaptation period the animals were divided into three groups of five pigs each. The first group served as control. The second group received 109 CFU EcN/d and the third group 1011 CFU EcN/d. EcN was given as a suspension in dilution medium (2.5 g NaCl, 2.5 g KCl, 0.2 g MgSO4, 0.2 g CaCl2, 0.2 g MgCl26H2O, 20 ml NaOH (32%) add distilled H2O to a total of 1000 ml) orally for 21 days. The control group received the dilution medium without EcN. The animals were killed by exsanguination following mechanical stunning. The abdominal contents were removed immediately after death. The protocol of animal treatment was approved and the procedure was supervised by an animal protection officer of the University.
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2.2. Fecal samples
2.4. Histology
To ensure proper administration of EcN, fecal samples were taken from every animal at days 7 and 14 of the experiment and after slaughter. They were analyzed for EcN content by multiplex PCR using strain-specific DNA primer sequences as described elsewhere (Blum-Oehler et al., 2003). All EcN-treated animals were tested positive for EcN at days 7 and 14 whereas control animals always yielded negative EcN RT-PCR results (data not shown).
Paraformaldehyde-fixed paraffin slides were deparaffinized, hydrated and stained using Meyer’s hemalum solution (Merck, Darmstadt, Germany) and aqueous eosin Y Solution (Sigma–Aldrich, Steinheim, Germany) according to standard protocols (Romeis, 1989). Carnoy’s solution fixed paraffin sections were stained with toluidine blue O (Sigma– Aldrich) for an hour and counterstained with Meyer’s haemalum. Following dehydration with increasing concentrations of alcohol and finally xylene, the slides were mounted in Entellan1 (Merck).
2.3. Tissue samples 2.5. Monoclonal antibodies Tissue samples were taken immediately after slaughter from the duodenum (10 cm aboral from the pylorus), jejunum (4.50 m aboral from the pylorus), ileum (5 cm oral from the ileo–colic orifice), ascending (10 cm aboral from the caeco– colic junction) and descending (60 cm aboral from the end of the ascending colon) colon. Mucosa preparations and small tissue specimen (0.5 cm) were immediately snap frozen in liquid nitrogen for RNA isolation and cryostat sectioning, respectively. An additional tissue specimen was collected at each location (0.5 cm) and fixed either in 4% paraformaldehyde at pH 7.2 for 18–24 h or in Carnoy’s solution (60% ethanol absolute, 30% chloroform, 10% glacial acetic acid) for 2 h. The samples were embedded in paraffin wax and sectioned at 2 mm, placed on SuperfrostTM plus slides (Menzel, Braunschweig, Germany) and dried overnight at 37 8C.
The antibodies used in this study are listed in Table 1. Biotinylated secondary Ab (sAb) were obtained from Southern Biotechnology, Birmingham, USA. 2.6. Immunohistochemistry Different immune cell populations were identified by immunohistochemistry using the enzyme-linked immunoperoxidase technique. Incubation steps were carried out in a humid chamber at room temperature if not indicated otherwise. After each step slides were washed for 5 min in TBS (pH 7.4) supplemented with 0.035% Tween120 (Sigma–Aldrich). CD4+ and CD8+ subpopulation analysis was performed using cryostat sections of 5 mm thickness placed on Superfrost1 plus glass slides (Menzel-Glaeser, Braunschweig, Germany). Sections were air-dried
Table 1 Antibody dilutions Primary Ab CD4 CD8 CD25 IgA Mouse IgG1 Mouse IgG2a Mouse IgG2b a b
Clone 74-12-4 MIL-12 231.3B2 K61 1B4 – – –
Company VMRD, USA Serotec, UK Serotec, UK Serotec, UK Dianova, Germany Dianova, Germany Dianova, Germany
Dilutions were prepared with Monet Blue (Biocare, Germany). Dilutions were prepared with VanGogh Yellow (Biocare, Germany).
Dilution a
1/50 1/100 b 1/20a 1/10a 1/20a 1/100 c 1/50b
Secondary Ab Goat–anti-mouse IgG2b Goat–anti-mouse IgG2a Goat–anti-mouse IgG1 Goat–anti-mouse IgG1 Goat–anti- mouse IgG1 Goat–anti-mouse IgG2a Goat–anti-mouse IgG2b
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for 30 min and fixed in a mix of equal amounts of acetone and methanol at 4 8C. Tissues were blocked with 5% normal swine serum diluted in Tris-buffered saline (TBS) for 30 min to reduce unspecific Abbinding, and then incubated overnight with the primary Ab at 4 8C. The following day slides were blocked again with 5% normal swine serum diluted in TBS, and then incubated with a biotinylated secondary Ab (1/50 dilution) for 30 min. Streptavidin–horse radish peroxidase (Biocare, Hamburg, Germany) and 3-amino-9-ethyl carbazol (AEC) were used to visualize specific binding of the Ab (Zymed, San Francisco, CA). Slides were counterstained with Meyer’s hemalum solution for improved cell differentiation and finally mounted in Faramount1 Aqueous Mounting Medium (Dako, Hamburg, Germany). Each staining series was accompanied by one slide treated with an isotype control replacing the primary Ab. Because of the lack of endogenous peroxidase activity in the cryostat sections, blocking with hydrogen peroxide was not necessary using this staining protocol. For the detection of CD25 and IgA paraffin embedded specimen were used. Slides were deparaffinized and hydrated. To ensure antigen de-masking the samples were treated with Dako1 Target Retrival Solution (pH 6) (Dako) in a water bath at 95 8C. The subsequent immunohistochemistry was performed as described above; however, an additional incubation in methanol containing 1.2% hydrogen peroxide was added following the incubation with the sAb to ensure quenching of endogenous peroxidase. 2.7. Cell counts Cells counts within the intestinal mucosa were performed at 1000-fold magnification using a light microscope Labophot-2 (Nikon, Tokyo, Japan). Cells were counted separately in the epithelium and the lamina propria (LP). Data were expressed in percent of positive cells per total number of epithelial cells or total number of cells in the LP, respectively. Cells were assigned to the intraepithelial compartment only if they were located with the epithelial cell band or at least half of their plasma membrane was in contact with the basement membrane of the epithelium. For CD8+ cells, we differentiated between luminal and basal section of the mucosa. Therefore, the luminal
compartment of the mucosa was considered to be the villi in the small intestine and the most luminal microscopic field of view (1000 times magnification) in the large intestine. The basal compartment of the mucosa contained the crypts in the small intestine and the most basal microscopic field of view in the large intestine. The assignment made in the large intestine ensured that cells were not accidentally counted for both compartments. CD4+ cells could be evaluated on a semi-quantitative basis only by assignment to four groups defined as follows: (1) <25% of LP cells CD4+; (2) 25–49% of LP cells CD4+; (3) 50–75% of LP cells CD4+; (4) >75% of LP cells CD4+. 2.8. RNA isolation and RT-PCR Total RNA was prepared from washed and stripped mucosa samples (0.9–0.8 g) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s manual. The amount of total RNA was quantified by spectrometry. For RT-PCR 200 ng of total RNA was used and processed as described previously (Gebhardt et al., 2002). Briefly, RNA was treated with 10 U RNase-free DNAse (Promega, Madison, WI) to remove genomic DNA. After denaturation at 70 8C cDNA was synthesized at 49 8C adding SuperscriptTM plus reverse transcriptase (Invitrogen, Karlsruhe, Germany) and oligo-dT primers (Pharmacia, Uppsala, Sweden). 1/10 of the total cDNA was used for one PCR reaction. PCR was performed with 30 s at 94 8C, 45 s at 55–58 8C, 35 s at 72 8C. Numbers of cycles and annealing temperatures differed for each pair of primers: IFN-gamma (34 cycles, annealing 58 8C), TNF-alpha (35 cycles, annealing 58 8C), TGF-beta (32 cycles, annealing 58 8C), IL-10 (40 cycles, annealing 58 8C) preprodefensin-beta 1 (39 cycles, annealing 58 8C), protegrins and PR-39 (42 cycles, annealing 55 8C), NKlysin (36 cycles, annealing 55 8C) and GAPDH (28 cycles, annealing 56 8C). To ensure that a saturation of primers were not misinterpreted as an increase of mRNA, as many cycles of PCR were used as were necessary to ensure the amplification reaction was still in its exponential phase. The number of cycles for every primer pair was determined by preliminary experiments with samples from at least three different untreated pigs and for every intestinal location tested. The cycle number was than used in treated and
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Table 2 PCR-primer
IFN-gamma TNF-alpha TGF-beta IL-10 PR-39 NK-lysin Protegrines Prepro-defensine-beta GAPDH
Sense-primer
Antisense-primer
50 -GCAAGTACCTCAGATGTACC-30 50 -ACTGCACTTCGAGGTTATCG-30 50 -CAGAGAGGCTATAGAGGGTT-30 50 -TTGCCAAGCCTTGTCAGAGA-30 50 -ACCCATCCATTCACTCAC-30 50 -GAGCAGTTCTGCCAGAACCT-30 50 -TGGATCAGATCAAGGACC-30 50 -CCTCCTTGTATTCCTCCTCA-30 50 -ACCACAGTCCATGCCATCAC-30
50 -TGGCCTTGGAACATAGTCTG-30 50 -AGAGGACCTGGGAGTAGATG-30 50 -TGTCTAGGCTCCAGATGTAG-30 50 -TCACCCATGGCTTTGTAGAC-30 50 -AGCCACAACAATAAGATCC-30 50 -GCAGGAGTTAGGTGAGAGAA-30 50 -ACACAGACGCAGAACCTAC-30 50 -TTGCAGCATTTGACTTGGGG-30 50 -TCCACCACCCTGTTGCTGTA-30
untreated animals to allow comparative semi-quantitative detection of mRNA expression. PCR was performed with 2.5 U Taq DNA polymerase (Invitrogen) and 20 pmol of specific sense and antisense primers shown in Table 2. The PCR products contained cDNA fragments of 359 bp (IFN-gamma), 266 bp (TNF-alpha), 371 bp (TGF-beta), 243 bp (IL-10), 168 bp (prepro-defensin-beta 1), 262 bp (PR-39), 100 bp (protegrins), 479 bp (NK-lysin) and 452 bp (GAPDH), respectively. Eleven microliters of the PCR products were visualized on 1% agarose gel containing ethidium bromide (500 ng/ml) and photographed.
significantly different when comparing small and large intestinal sites, their density within the LP ranging from 0.4% in the jejunum to 3.7% in the duodenum and colon (Fig. 1B). Mast cells were equally distributed throughout the intestinal LP, and accounted for about 3% of LP cells at all
2.9. Statistics Data were expressed as mean (S.D.) if not indicated otherwise. Differences in cell counts were accessed using the Student’s t-test and differences between experimental groups by one-way ANOVA.
3. Results 3.1. Number and distribution of granulocytes and mast cells In untreated animals, granulocytes and mast cells were detected in the lamina propria of the mucosa, but not in the epithelium. Eosinophils were concentrated within the basal part of the LP. They were much more frequent in the small compared to the large intestine, with highest numbers in the mucosa of the ileum (11 2.2% of total LP cells). In the colon almost no eosinophils were visible (0.6 0.3% of total LP cells) (Fig. 1A). Neutrophil numbers were not
Fig. 1. Eosinophil (A), neutrophil (B) and mast cell (C) density in the intestinal lamina propria mucosae (LP) shown as per cent of all lamina propria cells. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). Bars show the mean of five pigs per group with error bars indicating the standard deviation.
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investigated sites of the gut (Fig. 1C). No basophils could be detected in the intestinal mucosa of the pigs. The numbers of the three cell types examined here did not change following treatment of the animals with either low or high dose of EcN, as shown in Fig. 1. 3.2. Number and distribution of T lymphocyte subsets and plasma cells IgA producing plasma cells, CD4+ cells, CD25+ cells and CD8+ cells were analyzed by immunohistochemistry. IgA plasma cells were predominantly found in the LP of the crypts. They were virtually absent in the epithelium and in submucosal layers. Number of IgA plasma cells were highest in the duodenum with a density of 21.1% (3.1% of LP cells). Plasma cell counts were significantly lower at
Fig. 2. Density of IgA+ plasma cell (A), CD4+ cells (B) and CD25+ cells (C) in the intestinal lamina propria mucosae (LP) shown as per cent of all lamina propria cells. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). Bars show the mean of five pigs per group with error bars indicating the standard deviation.
the more caudal sites of the GI tract (jejunum, ileum, colon), where only 3% of LP cells were IgA plasma cells. The density of IgA plasma cells was not changed by treatment of the animals with EcN (Fig. 2A). Cells expressing the CD4 co-receptor were frequently found in the LP but were absent in the epithelium. Within the LP 50–75% of cells were CD4 positive. Therefore, we only performed a semiquantitative analysis. CD4+ cells were evenly distributed throughout cross-sections of the mucosa in the ileum and colon; however, the mucosa of the duodenum and jejunum contained higher numbers of CD4+ cells in the LP of the villi compared to the crypts (Fig. 2B). CD25+ cells were localized in small amounts (2.4–3.7% of total LP cells) in the intestinal mucosa. They were evenly distributed in the LP (Fig. 2C) and only sporadically detected in the epithelium. Again, neither CD4+ cell density nor CD25+ cell density changed following EcN treatment (Fig. 2B and C). CD8+ cells were the only ones found to be consistently present within the epithelium and the LP of the intestinal mucosa. The density of CD8+ cell was higher in the luminal compared to the basal
Fig. 3. Density of CD8+ cells in the luminal compartment of the mucosa (A) and the basal compartment of the mucosa (B), shown in percent of all epithelial cells, LP cells, respectively. Animals were either untreated (white bars), or treated with 109 (hatched bars) or 1011 CFU/d (black bars) of E. coli Nissle 1917 (EcN). First bar of every colour pattern indicating cells in the epithelium and the second bar cells in the LP. Bars show the mean of five pigs per group with error bars indicating the standard deviation. *p < 0.05 compared to same intestinal segment in the control group.
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compartment of the mucosa. Highest counts were found in the luminal epithelium of the duodenum. Along the intestinal mucosa the CD8+ cell counts in the LP were reverse to those in the epithelium of the same compartment. For example in the luminal compartment of the mucosa the epithelium contained much higher numbers of CD8+ cells than the corresponding LP (Fig. 3A). In the basal compartment of the mucosa the opposite was true (Fig. 3B). Animals receiving high amounts of EcN
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(1011 CFU/d) showed significantly higher CD8+ cells counts in the luminal LP of the Ileum, in the epithelium and the lamina propria of the ascending colon compared to placebo-fed pigs (Figs. 3 and 4). In low dose fed animals CD8+ cells where increased in the basal epithelium of the jejunum and in the luminal LP of the ascending colon (Fig. 3). No significant changes in CD8+ cell counts were observed in the mucosa of other intestinal segments on EcN treatment.
Fig. 4. Pictures showing cells bearing the CD8 antigen (red) in the mucosa of the ascending colon. The animal treated with 1011 CFU/d (B) of EcN shows an increase in the number of CD8 positive cells compared to the untreated animal (A). Slides were stained by immunohistochemistry using the MIL-12 Ab (Serotec, UK) or a mouse IgG2a (Dianova, Germany) isotype as negative control (C). Pictures from one representative animal per group are shown. Metering bar = 25 mm.
Fig. 5. mRNA expression of cytokines (IFN-gamma, TNF-alpha, TGF-beta, IL-10) and antimicrobial peptides (prepro-defensin-beta 1, NKlysin) in the ileum and the ascending colon compared to housekeeping gene (GAPDH). Pictures show two representative animals of the control group and the group treated with 1011 CFU/d of E. coli Nissle 1917 (EcN). Numbers indicate the PCR cycles accomplished.
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3.3. mRNA expression of different cytokines and AMP Total RNA from intestinal mucosa of duodenum, ileum and ascending colon in control animals and animals fed 1011 CFU EcN/d was investigated. mRNA expression of different cytokines and antimicrobial peptides was evaluated semi-quantitatively by comparison to GAPDH as housekeeping gene to check for possible changes in immune cell subpopulations. mRNA expression of IFN-gamma, TNFalpha, TGF-beta and IL-10 as well as the mRNA expression of the AMP: prepro-defensine-beta 1 and NK-lysin was detected in the duodenum (data not shown), ileum and ascending colon (Fig. 5). In all samples PR-39 was only partly seen in the intestinal mucosa and mRNA for protegrins was absent in all animals and intestinal segments (data not shown). Transmural duodenal tissue samples of 3-week-old piglets were included as positive controls. Neither in the investigated cytokines nor in the AMP changes were detected after feeding high amounts of EcN (1011 CFU/d).
4. Discussion During the last decades pigs health became a huge economic factor in livestock farming because of intensive animal husbandry and increasing production rates. Simultaneously, the opportunities of prophylactic treatment against the major porcine pathogens were limited due to governmental restriction (prohibition of antibiotics in animal feed, etc.). Therefore, alternatives are needed. Probiotics showed the potential to fulfil such requirements, even though the mechanisms of their beneficial effects are mostly unknown. This study was performed to investigate the effects of EcN, a probiotic E coli strain successfully used in human and veterinary medicine, on the intestinal immune system of healthy young pigs. The aim was to further understand the modes of action of this probiotic strain, which obviously has beneficial properties both in man (Kruis et al., 2004) and in calves (von Buenau et al., 2005). We selected the pig as a model not only because this species might as well benefit of probiotic treatment (Alexopoulos et al., 2004), but also because the pig intestinal immune
system is considered to be much closer to the human one than the rodent immune system (Miller and Ullrey, 1987; Boes and Helwigh, 2000). Studies have been performed in pigs on the distribution of particular immune cells in the blood and in different tissues (Bianchi et al., 1992; Yang and Parkhouse, 1996; Boeker et al., 1999; Saalmuller et al., 2002; Sinkora et al., 2005) including studies on T cells in the intestine (Vega-Lopez et al., 1993; Pabst and Rothko¨tter, 1999). Nevertheless, it is surprising to note that no published data are available on the distribution of cells of the innate immune system within the intestine of the pig. Therefore, we were interested not only in studying the influence of EcN on intestinal immune cells, but also in quantifying the number and distribution of granulocytes, mast cells and lymphocyte subsets in the gut mucosa. Eosinophils were mainly found in the LP of the crypts in the small intestine and the basal part of the crypts in the large intestine, as described previously (Bianchi et al., 1992). Eosinophils could be found at all sites of the small and large intestine, the highest numbers being seen in the ileum. Interestingly, we found for the first time that the density of eosinophils in the colon LP was lower than 1% and thus much lower than that described in humans (Bischoff et al., 1996). Because none of the animals showed signs of parasite infection and all were dewormed on a routine schedule, an increase of eosinophils in the small intestine due to parasitosis can be largely ruled out. There is evidence from the literature that gastrointestinal eosinophils not only act as inflammatory effector cells of allergy and parasitic infection, but are also capable of functioning as immunomodulating cells (Kato et al., 1998). Kato et al. showed a spontaneous minimal degranulation of intestinal eosinophils in the healthy gut that does not occur in other normal tissues. These data suggest that eosinophils may also act as immune-modulating cells in the small intestine of pigs. Compared to eosinophils the amount of neutrophils in the intestinal mucosa of healthy pigs was much lower. Neutrophil counts were highest in the duodenum and descending colon. The amount of neutrophils was higher than the numbers regarded to be normal in humans (0.4%) (Ogra et al., 1999). In the duodenum the reason might be that pigs are more prone to contact with microorganisms because
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of their usual environment. Therefore, more neutrophils may ensure a fast reaction to potential pathogens. The relatively high amount of neutrophils in the colon, compared to humans, is likely to be a result of higher physiologic amounts of bacteria in this part of the pig intestine compared to the human colon. In contrast to neutrophils, the counts of mast cells were in the expected ranges, since our data were similar to those reported by Xu et al. (1993) who did similar studies in pigs. Mast cells, unlike granulocytes, were evenly distributed throughout the intestinal segments. Treatment with EcN had no effect on the number and distribution of granulocytes and mast cells in healthy pigs. Moreover, the absence of major changes in tested cytokine and AMP mRNA expression makes an influence of EcN on the cellular metabolism unlikely too. Even though, a definite conclusion on cytokine-mRNA concentration would require a quantitative method for mRNA detection like real time PCR, because the semi-quantitative method used can only detect major changes. However, the unaltered number of cells in healthy EcN-treated animals does not rule out a positive effect on disease-induced cell recruitment (Sturm et al., 2005; Kamada et al., 2005). From experiments with other probiotics like lactobacilli it is known, that they are able to decrease infection-induced, transmucosal migration of neutrophils (Michail and Abernathy, 2002). Keeping in mind EcN being a probiotic, the fact that it does not recruit neutrophils in healthy animals rather underlines its apathogenicity than its ineffectiveness. Even though in the present study no changes were detected with respect to the number and distribution of intestinal eosinophils, it still needs to be clarified if the administration of EcN leads to alterations in mediator composition and release in this cells. In accordance with findings of other authors (Rothko¨tter et al., 1991; Bianchi et al., 1992) IgAproducing plasma cells were mainly found in the basal part of the mucosa with highest amounts in the crypts of the duodenum. No changes were detected following EcN treatment. An increase in the number of intestinal IgA producing plasma cells, does not seem to be a mechanism of action of EcN in conventionally reared, healthy pigs. Nevertheless, unchanged IgA plasma cell counts do not rule out an influence of EcN on the luminal secretory IgA concentration as reported for
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other probiotics like Saccharomyces boulardii (Buts et al., 1990; Rodrigues et al., 2000). Heterogeneous results were obtained with regard to lymphocyte subsets. We found no changes in CD4+ and CD25+ cells, but an increase of CD8+ cells in the ascending colon induced by EcN. In contrast to the results of others (Vega-Lopez et al., 1993), we found in our experiments polarization of mucosal CD4+ cells towards the LP of the villi only in the duodenum and jejunum. In the ileum and colon, the CD4+ lymphocytes were equally distributed throughout the mucosa. An explanation of this difference might be the lower age of the animals used in the study by Vega-Lopez et al. Even though no alterations in the CD4+ cell counts were evident between treatment and control groups, an influence EcN on the balance of CD4+ subpopulations cannot be ruled out. Different from CD4+ cells significantly more CD8+ cells were counted in the luminal epithelium than in the lamina propria of the intestine, as reported earlier by other groups (Vega-Lopez et al., 1993, 2001; Pabst and Rothko¨tter, 1999). This holds true for all intestinal segments investigated. We extended these findings by distinguishing between the epithelium and the LP in the luminal and the basal part of the mucosa, respectively. The highest number of CD8+ cells (one-third of total cell count) was found in the luminal epithelium of the duodenum. Different from the findings of Vega-Lopez et al. we could not confirm that the CD8+ cells of the LP were localized near the basement membrane of the epithelium, possibly because of different methodological approaches. We report here for the first time that EcN treatment increased the amount of CD8+ cells in the ascending colon in healthy young pigs. Feeding 1011 CFU EcN/d for 21 days resulted in an increase of CD8+ cells in the ascending colon, both in the luminal as well as in the basal compartment of the mucosa. In the absence of mucosal inflammation (no increase in neutrophils, no differences of proinflammatory cytokine-mRNA expression in comparison between treated and untreated groups, no activation of lymphocytes) the high numbers of CD8+ cells cannot be interpreted as an inflammatory response. From our experiments it cannot be concluded whether the increased number of CD8+ cells detected in the epithelium of the ascending colon is due to proliferation of CD8+ cells or to recruitment of cells from other sites like the lamina
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propria or the mesenteric lymph nodes. However, it has been shown in mice that intestinal IEL are able to proliferate in the epithelium (Lin et al., 1994). The accumulation of lymphocytes originating from the peripheral blood as an explanation of the increase of CD8+ cells due to EcN treatment is unlikely, since Sturm et al. (2005) showed a down-regulation of proliferation in peripheral blood T cells by EcN in vitro, whereas the proliferation of LP T cells was unchanged. The cell population expressing CD8 coreceptor in the intestine consists of different cell types including cytotoxic T cells, gamma/delta T cells and NK cells. In this study we did not differentiate between subtypes of CD8+ cells as CD8high and CD8low. Therefore, no conclusion can be drawn on changes within a special subtype of CD8+ cells; however, the beneficial effects of probiotics such as EcN could be related to known effects of CD8+ subpopulations, such as defence against pathogens by cytotoxic CD8+ cells, or maintenance and enhancement of the epithelial integrity by gamma/delta CD8+ cells (Chen et al., 2002). Other than in humans and mice intraepithelial CD8+ gamma/delta T cells have not yet been identified in the intestine of pigs, but that does not rule out the possibility that equivalent subtypes of CD8+ cells exist also in pigs within the epithelium. Especially, because there is increasing evidence that pigs have a distinct repertoire of intestinal gamma/delta TCR (Holtmeier et al., 2002) and T cells expressing the delta chain of the TCR have been shown in the colonic epithelium (Hontecillas et al., 2005). Future experiments are needed to further characterize the phenotype of CD8+ cells in the pig, and to establish their biological relevance in disease and disease prevention. Even though the porcine intestinal immune system might be an excellent model in terms of transfer of results to the situation in humans, there are of course some limitations. The lack of EcN effects on the density of innate immune cells such as granulocytes or mast cells shown in this study, as well as on the density of CD4+ cells (mostly T helper lymphocytes), CD25+ (activated T cells) and IgA cells might be the result of the limited number of animals examined here. Unfortunately, the repertoire of antibodies directed against porcine cell surface antigens is limited compared to that available for rodents. A future improvement in availability of
directly fluorescent-labeled antibodies to perform fluorescent immunohistochemistry and an improvement in isolation techniques for intestinal immune cells will be important to defining specialized subsets of lymphocytes and innate immune cells in situ. This will help to further investigate an influence of EcN on intestinal immune cells in the pig. Furthermore, using EcN in an infection model might be useful to reveal changes in the reaction and migration of intestinal immune cell population that cannot be detected in healthy animals.
5. Conclusions We showed in this study as described earlier in humans (Kruis et al., 2004) and mice (Waidmann et al., 2003; Schultz et al., 2004), that EcN did not induce any signs of inflammation and did not exhibit any signs of pathogenicity, even when fed to young pigs at high doses. The EcN-induced enhancement of the number of CD8+ cells in the ascending colon might be linked to the beneficial role of maintaining porcine intestinal health and preventing disease. Intestinal epithelial cells have been shown in vitro to produce keratinocyte growth factor (KGF) (Chen et al., 2002) and might thereby increase the epithelial integrity. Mice lacking gamma/delta TCR IEL have impaired epithelial development in the intestine (Boismenu and Havran, 1994). It has also been shown recently that IEL express junctional molecules like epithelial cells, whereas lymphocytes from other body sites (spleen, mesenteric lymph nodes, thymus, Payer’s patches) do not (Inagaki-Ohara et al., 2005). This opens up new possibilities for the involvement of IEL in the epithelial barrier and for the communication with epithelial cells. To further evaluate the beneficial effects of EcN in pigs and the underlying mechanisms, experiments with pig infection models are of special interest.
Acknowledgements We thank Gisela Weier and Gerhild Becker for excellent technical assistance and Yvonne Armbrecht and Michael Rohde for help with the animals as well as Anne Luschert for advice in immunohistochem-
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