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Am J Clin Nutr 89: 871-879, 2009. First published January 28, 2009; doi:10.3945/ajcn.2008.27073
 American Journal of Clinical Nutrition, doi:10.3945/ajcn.2008.27073
Vol. 89, No. 3, 871-879, March 2009
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© 2009 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Generation of epidermal growth factor–expressing Lactococcus lactis and its enhancement on intestinal development and growth of early-weaned mice

Queenie CK Cheung1,2,3, Zongfei Yuan1,2,3, Paul W Dyce1,2,3, De Wu1,2,3, Kees DeLange1,2,3 and Julang Li1,2,3

1 From the Department of Animal and Poultry Science, University of Guelph, Guelph, Canada (QCKC, ZY, PWD, KD, and JL), and the Institute of Animal Nutrition, Sichuan Agricultural University, Ya-an, China (DW).

2 Supported by Ontario Pork, Natural Sciences and Engineering Research Council, and Agriculture and Agri-Food Canada. QCKC was the recipient of the Vitamin Doctoral Research Scholarship provided by the University of Guelph.

3 Reprints not available. Address correspondence to J Li, Department of Animal and Poultry Science, University of Guelph, Guelph N1G 2W1, Ontario, Canada. E-mail: jli{at}uoguelph.ca.


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ABSTRACT
 
Background: Epidermal growth factor (EGF) plays an important role in intestinal proliferation and differentiation. Previous studies by others have shown that administration of EGF into the ileum lumen enhances intestinal development.

Objective: The objective was to examine the feasibility of expressing and delivering EGF via Lactococcus lactis to early-weaned mice to enhance intestinal development at this critical transition stage.

Design: EGF-expressing L. lactis (EGF-LL) was generated with a recombinant approach. Early-weaned mice were orally gavaged with the recombinant bacteria. Body weight, mean villous height, and crypt depth in the intestine were measured to examine the influence of EGF-LL on the intestinal development of early-weaned mice in vivo.

Results: Populations of EGF-LL were shown to survive throughout the intestinal tract, and the recombinant EGF protein was also detected in intestinal contents. Weight gain was significantly greater in mice that received EGF-LL than in control mice fed phosphate-buffered saline or L. lactis transformed with the empty vector backbone but was comparable with that of the positive control mice that received recombinant human EGF. EGF-LL increased mean villous height and crypt depth in the intestine. Immunohistochemistry also confirmed that enterocyte proliferation was enhanced in mice that received EGF-LL, as evidenced by the greater number of cells stained with proliferative cell nuclear antigen in the intestine.

Conclusions: This study showed that EGF-LL had beneficial effects on the intestinal growth of newly weaned mice. The combination of growth factor delivery and a probiotic approach may offer possibilities for formulating dietary supplements for children during their weaning transition stage.


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INTRODUCTION
 
Disturbed digestive function and the associated reduction in growth rates during the period immediately after weaning is one of the major problems in nursery animal management (1). Animals experience stress during this period, largely because of changes in food composition and an immature gastrointestinal (GI) tract. This leads to damaged mucosal integrity and reduced nutrient digestion and absorption (2). As a result, more substrates are available for enteric pathogenic bacteria; therefore, animals at the weaning stage become more susceptible to infection and intestinal disorders. Developing a strategy to stimulate intestinal growth and development of early-weaned animals may help to optimize their performance during this critical stage.

Epidermal growth factor (EGF) is a 53-amino acid single-chain polypeptide with a molecular mass of 6045 Da that was first isolated from male mouse submandibular glands (3) but is now found in various tissues of all mammalian species examined (see reference 4 for review). Maternal colostrum and milk are the main sources of intestinal EGF during the postnatal stage, and it is known that the concentration of EGF in sow milk is 124 µg/L (5). In addition, EGF is also produced in the salivary glands (6, 7). Exogenous infusion of EGF in utero in rabbits has been shown to accelerate the maturation of intestinal enzyme activity as well as stimulate intestinal growth (8). It was also reported that administration of EGF through a catheter into the ileum lumen of adult rats significantly increases intestinal development, reflected by the increase in mean mucosal ornithine decarboxylase–specific activity, crypt labeling index, and mean DNA content in the mucosa of the ileum (9). In addition, in newborn and weaned piglets, systematic or oral administration of EGF significantly increased jejunal lactase and sucrase activities (5, 10), which suggests that EGF also modulates enterocyte differentiation.

The possibility of EGF administration on enhancing intestinal repair after damage has also been investigated. For example, it was shown that EGF reduced colonization of the intestinal epithelium by enteropathogens (11–13). Oral EGF administration inhibited enteropathogenic Escherichia coli (EPEC)–induced diarrhea and prevented a reduction in weight gain when administered prophylactically (11). Supplementation of formulas with EGF facilitated the recovery of the intestine in piglets from a rotavirus infection (14). Thus, the therapeutic potential of growth factors as a novel approach for intestinal development and infectious diarrhea control has been shown. However, recombinant protein production and purification with the use of conventional methods is very costly. A cost-effective approach to express and deliver recombinant growth factors to the intestine is crucial.

Lactococcus lactis is a nonpathogenic, noninvasive, noncolonizing, gram-positive lactic acid bacterium that is mainly used to produce fermented foods. Lactic acid–producing bacteria (LAB) are generally regarded as safe organisms and are therefore widely used in the production and preservation of fermented products by the food industry. L. lactis has been considered a good candidate to serve as a cost-effective live vaccine delivery vehicle for mucosal immunization. L. lactis is metabolically active in all compartments of the intestinal tract (15), which makes live delivery of recombinant protein to the intestine possible. The potential of L. lactis to express and secrete fully biologically active cytokines has been shown (16, 17). Steidler et al (18) had also constructed a thymidine-dependent human interleukin-10 (IL-10)–expressing L. lactis, which is unable to survive outside the GI tract and thus eliminates the risk of the genetically modified bacterium contaminating the environment. The current study tested the hypothesis that local delivery of EGF in the GI tract via L. lactis enhances intestinal development and integrity and is thus beneficial for improving the performance of early-weaned animals.


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MATERIALS AND METHODS
 
Cloning of the pAMJ399-EGF expression construct
Total RNA was isolated from pig salivary gland tissue by using the Trizol method according to the manufacturer's instruction. Reverse transcription and polymerase chain reactions were carried out by using forward (5'-GCTCCGAGATCTAATAGTTACTCTGAATGC-3') and reverse (5'-GAGAATCTGCAGCTAGCGCAGCTCCCACCATTT-3') primers to amplify the mature EGF sequence [GenBank NM_214020 (19); bold letters indicate the addition of restriction enzyme sites of BglII and PstI in the forward and reverse primers, respectively]. Polymerase chain reaction (PCR) conditions were 94°C (3 min) [94°C (45 s), 58°C (30 s), and 72°C (1.5 min)] for 35 cycles and 72°C (10 min) for the final extension. The primers, spanning exons 20 and 21, would amplify a 159-bp mature EGF DNA product that was verified by 1% agarose gel electrophoresis. The resulting PCR product was gel-purified by using a QIAEX Gel Extraction Kit (Qiagen, Germantown, MD) and ligated into pGEM-T Easy vector (Promega, Madison, WI) to generate pGEM-EGF. After transforming into E. coli DH5{alpha}, pGEM-EGF was digested with BglII and PstI to release the EGF insert. The purified EGF insert was subsequently cloned into the multiple cloning site (MCS) of a 6949-bp expression vector pAMJ399 (Bioneer, Hørsholm, Denmark; 20) that was linearized with BglII and PstI. The resultant construct, known as pAMJ399-EGF, contains a pH-regulated promoter, P170, a secretion signal peptide SP310mut2, a p15A replicon, and an erythromycin-resistance gene for selection. The expression construct was then transformed into L. lactis MG1363 and PSM565 competent cells via electroporation. DNA sequencing confirmed the identity of the plasmid. The original pAMJ399 vector without the EGF insert was also transformed into both strains of L. lactis to serve as controls and was designated as empty vector control (EV-LL).

Recombinant L. lactis growth and fermentation
Frozen inoculum stocks of EGF expressing L. lactis (EGF-LL) were stored in 20 mmol/L K-PO4 with 50% (vol:vol) glycerol at –80°C. Glycerol stocks of the recombinant L. lactis were streaked on M17 agar (Oxoid, Basingstoke, United Kingdom) plates, supplemented with 1% D-glucose and 1 µg/mL erythromycin (M17-G1-ery; Sigma-Aldrich, St Louis, MO), and grown at 30°C for 18 to 22 h. A single colony was inoculated in 10 mL M17-G1-ery medium and incubated at 30°C overnight without shaking. The growth of the recombinant L. lactis was studied at different time points from 16 to 72 h. The concentration of the cultures was determined by optical density at 600 nm (OD600), measured by spectrophotometry, and pH values were simultaneously measured with a digital pH meter (Fisher Scientific, Pittsburgh, PA). At each time point, 1 mL of the culture was centrifuged at 5000 rpm for 10 min at 4°C and both the supernatant fluid and cell pellet were stored separately at –80°C for later analyses.

Western blot analysis
Proteins in cell lysates and supernatant fluids were separated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes at 4°C for 1 h. The blots were blocked at room temperature for 1 h in 5% skim milk–phosphate-buffered saline (PBS) and then incubated with primary anti-EGF (1:500 dilution; Cell Sciences, Canton, MA) overnight at 4°C. Membranes were washed in PBS-0.2% Tween 20 and then blotted in anti-rabbit IgG horseradish peroxidase–linked antibody (1:2000 dilution; Cell Signaling Technology, Beverly, MA) at room temperature for 1 h. After washing, the ECL plus Western Blotting Detection System kit (Amersham, Piscataway, NJ) was used to visualize the EGF protein band according to the manufacturer's instructions. X-ray films were developed by using an SRX-101A film processor (Konica), and images of the resulting X-ray films were captured by using GeneSnap software.

Mouse fibroblast isolation and in vitro cell proliferation assay
Newborn mouse hearts were removed with a scalpel and minced into 1 mm3 explants for fibroblast cell harvest. Thoroughly rinsed explants were allowed to adhere to the bottom of 10-cm petri dishes and overlaid with 10 mL Dulbecco's modified Eagle's medium + antibiotics supplemented with 10% fetal bovine serum (FBS). Cultures were maintained at 37°C in an atmosphere of 10% carbon dioxide/air. The culture medium was changed every 4–5 d. The primary monolayer of cells was trypsinized (trypsin-EDTA) when confluent and split into 1:3 in subculturing. Cells of third to fourth passage were used for the proliferation assays, which occurred {approx}5 wk after the initial tissue harvest. Mouse fibroblast cells at passage 3 or 4 were seeded in 6-cm dishes at an initial cell density of 0.35 x 105 cells and incubated until 70% confluent. Culture medium was aspirated by vacuum, and attached cells were washed once by sterile 1x PBS. The cells were then incubated in Dulbecco's modified Eagle's medium only for 24 h to achieve serum deprivation. After 24 h of FBS deprivation, cells were treated with the following groups: recombinant human EGF (rhEGF; 20 ng/mL), supernatant fluid collected from pAMJ399 empty vector transformed L. lactis culture at 24 h of fermentation (EV-LL; 15 µL) and supernatant fluid collected from the pAMJ399-EGF–transformed L. lactis culture at 24 h of fermentation (EGF-LL; 10 or 15 µL). The EV-LL and EGF-LL samples were filtered by using 0.45-µm syringe filters before treatment to the cells. Twenty-four hours after the cells were treated with their respective treatments, cells were trypsinized and quantified in a hemocytometer chamber. Cell counting was done in a blinded-manner, for which results represent the mean data collected by 2 individuals who were not aware of the experimental treatments.

5-Bromodeoxyuridine (BrdU) incorporation assays were also performed to determine the percentage of cells at the S phase of the cell cycle. Fibroblasts were cultured with the treatments described above, and BrdU (10 µg/mL; Sigma-Aldrich, St Louis, MO) was added to the culture 12 h before the end of the culture. Cells were then fixed in 4% formaldehyde diluted in PBS for 30 min at room temperature and treated with 0.1 mol glycine for 20 min and with 0.5% HCl and 0.1% Triton X-100 for an additional 45 min at room temperature to allow permeabilization. After being washed in PBS and buffered in Hank's Balanced Salt Solution for 3 min, the cells were incubated with anti-BrdU monoclonal antibody [Sigma-Aldrich; 1:50 in blocking buffer: PBS, 0.5% bovine serum albumin (BSA), and 0.5% Tween 20] overnight at 4°C. Cells were then incubated in secondary goat anti-mouse IgG (1:100) for 1 h at room temperature and washed, and 10 µg/mL propidium iodide suspended in PBS was added to the cells for 10 min of incubation to stain the nucleus. Cells were then washed, mounted on coverslips, and stored at 4°C until evaluation by fluorescent microscopy. The percentage of BrdU-positive cells was calculated by counting ≥5 random views of the BrdU-positive cells (green) out of nuclear-stained cells (red). The final percentage of each group represents the mean ± SD of 3 experiments.

Oral administration of recombinant EGF-expressing L. lactis
The procedures for use of animals in this study were in accordance with the guidelines of the Canadian Council for Animal Care Guidelines, and all work was approved by the University of Guelph Animal Care Committee. Adult CD-1 mice (6–7 wk) were purchased from Charles River (Wilmington, MA), and 3 separate breeding pairs were set up to generate pups for experiments. Weaning mice at 19–21 d of age were withdrawn from the parental cage and were randomly assigned to one of the following groups: rhEGF, EV-LL, EGF-LL, and 1x PBS. All mice were housed in a temperature-controlled environment with a 12-h light and 12-h dark cycle and provided free access to water and 14% rodent diets (Harlan Teklad Global Diets) Each individual mouse was tagged with identification, and initial body weight (BW) was recorded. Mice were orally fed, via intragastric gavage, twice daily through sterile animal feeding needles (Popper & Sons, New Hyde Park, NY) every morning and late afternoon for 9 consecutive days. For each feeding, mice received a dose of 300 µL fresh bacterial culture of EV-LL (13 mice) or EGF-LL (16 mice). The rhEGF group (11 mice) received 50 µg/kg of the recombinant human EGF daily at the same volume (300 µL). Negative control mice were given 300 µL sterile PBS (10 mice) only. A total of 50 mice were used in this study, and a maximum of 4 mice were housed in each cage within the same treatment group. Bacterial concentration in the fed culture was determined by plating serial diluted cultures on M17-G1-ery plates, enumerated colonies were expressed in CFU/mL. The mean EGF-LL and EV-LL concentrations were 2.71 ± 0.12 x 109 CFU/mL and 2.33 ± 0.08 x 109 colony-forming units (CFU)/mL, respectively. BW and food intake were recorded every 2 d, and signs of diarrhea, sickness, or abnormal behavior were also monitored throughout the experimental period. Final BW change was calculated as a percentage of the initial BW of each mouse. All mice were killed on day 9 by cervical dislocation, and tissue samples were collected.

Detection of EGF-LL in intestinal contents
To determine the survival of the recombinant L. lactis in the mouse guts, intestinal contents were collected into sterile 1.5-mL tubes and immediately serial-diluted for plating. Briefly, samples were weighed, and 100 µL of 1x sterile PBS was added to facilitate homogenization with an automatic homogenizer; 100 µL of each diluted sample was plated on M17-G1-ery plates and incubated at 30°C for 24 h. For each animal sample, 10 single recovered colonies were randomly chosen and used directly for PCR-colony screening according to previously established methods (21). PCR was performed by using P170 forward primer (5'-CTGCCTCCTCTCCCTAGTGC-3'), which anneals to the P170 promoter region, and reverse primer (5'-CTAAGGATGATTTCTGGCAGGG-3'), which anneals to the transcriptional terminator. PCR amplification is expected to result in a 453-bp product with EGF insert, or a 303-bp product with pAMJ399 plasmid vector only, or no product if plasmid was not present. PCR conditions were as follows: 94°C (10 min) [94°C (1 min), 55°C (1 min), and 72°C (2 min)] for 30 cycles and 65°C (2 min).

Histologic examination of intestinal morphology
Approximately 0.5 cm of the second centimeter of each small intestinal section (duodenum, jejunum, and ileum) was isolated, rinsed with sterile PBS, and fixed overnight with 10% formalin. Fixed tissues were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin (H&E) for morphologic examinations. The identities of all tissue slides were disguised; thus, an examination was conducted in a blinded manner. For each individual mouse, 3 cross sections of each of the small intestinal segments were reviewed. In each cross-sectioned tissue sample, ≥15–18 complete villous-crypt structures were examined under a microscope, and lengths of villous height and crypt depth were measured by using OpenLab software. All data for each animal were first averaged as a single mean of each segment per animal and further averaged as a group for each treatment.

Immunohistochemistry of proliferating cell nuclear antigen
The immunohistochemistry protocol used was adapted from a published method with slight modification (22). Briefly, paraffin-embedded tissue slides (0.5 µmol/L) were first deparaffinized by soaking in 100% xylene and rehydrated with a decreasing percentage of ethanol. Slides were then incubated in a bath of 10 mmol/L sodium citrate (pH 6.0) at 85°C for 1 h for antigen retrieval and transferred to a bath containing 0.2% sodium borohydride to block endogenous peroxides. After being washed 3 times with PBS, the slides were blocked in blocking buffer containing 5% BSA, 2% FBS, and 0.025% Triton X-100 in PBS at room temperature for 1 h. Slides were incubated in mouse monoclonal-proliferating cell nuclear antigen (PCNA) antibody (Abcam, Cambridge, MA) diluted in blocking buffer (1:2000) overnight at 4°C in humidified chambers. After the primary antibody was rinsed off, the slides were then incubated in biotin-conjugated goat anti-mouse immunoglobulins (1:200; Abcam) for 2 h at room temperature. All slides were treated with horseradish peroxidase–conjugated streptavidin (DakoCytomation, Glostrup, Denmark) for 25 min and reacted with 1 mL 3,3-diamino-benzidine tetrahydrochloride (Biochain, CA) as the colored chromogen for visualization of the antigenic structures. Slides were then counterstained in Harris' hematoxylin, mounted by using liquid mounting medium with coverslips, and examined under the bright field of light microscopy at both 10x and 40x magnifications. Incubation of successive tissue sections with secondary antibody only served as a negative control.

Statistical analysis
All experiments were performed 3 times, and the results are expressed as means ± SEMs. The data were analyzed by one-factor analysis of variance (ANOVA) using Prism version 3.03 analysis software (La Jolla, CA). Data sets were further analyzed by using Tukey's test for multiple comparisons to determine statistical differences between groups. The results were considered significant at a P value of <0.05.


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RESULTS
 
Generation of EGF-expressing recombinant L. lactis
EGF cDNA was cloned into pAMJ399 to generate pAMJ399-EGF. After the sequence identity was confirmed, pAMJ399-EGF was transformed into both MG1363 and PSM565 host strains. The growth curve and pH dynamic changes during a 72-h fermentation period are shown in Figure 1A. The peak growth of the recombinant bacteria occurred at 24 h, as evidenced by OD600 values of 3.5 and 3.3 for the MG1363 and PSM565 strains, respectively. The pH of the initial culture was {approx}6.9, which decreased to 5.25–5.26 at 24 h, which indicated active fermentation. The decrease in pH to <6 should have activated the expression of EGF driven by the active P170 promoter. Because no significant difference was observed between the 2 lines of recombinant L. lactis, subsequent experiments were performed with the MG1363 strain, designated as EGF-LL. To investigate whether the recombinant L. lactis are capable of producing EGF, Western blot analyses were performed by using a specific antibody against EGF. As shown in Figure 1B, EGF protein ({approx}6 kDa) was detected in both of the bacterial cell lysates and culture supernatant fluid, which suggested that the growth factor is produced and secreted by the recombinant L. lactis. By comparing the intensities of bands derived from the purified recombinant hEGF protein standards with those of the bands from the supernatant fluid samples, we determined that {approx}500 ng EGF was present per milliliter of the bacterial culture (Figure 1C).


Figure 1
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  		FIGURE 1. Fermentation of epidermal growth factor–expressing Lactococcus lactis (LL-EGF) and synthesis of recombinant EGF (rhEGF). (A) Comparison of the in vitro growth of recombinant L. lactis [MG1363 and PSM565 strains (MG-EGF and PSM-EGF, respectively)]. Optical densities at 600 nm (OD600) showed that both strains bearing the same pAMJ399-EGF plasmid achieved similar growth characteristics in a 72-h fermentation study. The pH values of the culture remained at 5.2–5.3 from 16 to 72 h. (B) Western blot analyses using polyclonal human EGF antibody showed that the 6-kDa EGF protein was present in both the cell lysates and supernatant fluid of the fermentation cultures at 24, 40, 48, 64, and 72 h. (C) Comparison of the intensities of rhEGF with EGF produced by recombinant L. lactis by Western blot. Known concentrations of rhEGF were loaded on the same sodium dodecyl sulfate–polyacrylamide gel electrophoresis to estimate the approximate production of the recombinant protein by LL-EGF, for which 45 µL of the supernatant fluid was loaded per well.

EGF protein produced by recombinant L. lactis was functional in vitro
To study whether the EGF expressed and secreted by the EGF-LL is indeed functional, cell proliferation assays were performed. Mouse fibroblasts were cultured in the absence and presence of spent supernatant fluid from EGF-LL culture for 24 h. Cells were then trypsinized and enumerated by using a hemocytometer. As shown in Figure 2A, the proliferation of mouse fibroblasts was significantly stimulated by 10 µL of the supernatant fluid from EGF-LL culture (1.16 x 106 ± 0.03 cells), when compared with the control group, in which fibroblasts were cultured in the presence of supernatant fluid from pAMJ-empty vector–bearing L. lactis culture (0.42 x 106 ± 0.04 cells; P < 0.05). The stimulating effect of EGF-LL was comparable with that of 20 ng/mL of recombinant human EGF (1.25 x 106 ± 0.09 cells; P > 0.05). The mitogenic effect of EGF produced by L. lactis was confirmed by a BrdU incorporation assay with mouse fibroblast cells. Green (BrdU positive) and red (nuclear positive) signals were quantified and expressed as a percentage of DNA synthesizing cells (green/red x 100%). The cells that received 10 µL EGF-LL resulted in significantly higher BrdU-positive stained cells (22.5 ± 3.8%) than did the EV-LL cells (9.38 ± 1.04%), which indicated that more cells had undergone DNA synthesis (Figure 2B). There was no difference between the EGF-LL and rhEGF treatment groups (21.4 ± 3.9%). Treatment of cells with 15 µL EGF-LL did not further increase the percentage of Brdu-positive cells.


Figure 2
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  		FIGURE 2. Epidermal growth factor (EGF) secreted by EGF-expressing Lactococcus lactis (EGF-LL) was functional. (A) Fibroblast cells were treated in supernatant fluid of EGF-LL, L. lactis that was transformed with the empty vector backbone (EV-LL), and recombinant human EGF (rhEGF) for 24 h and then trypsinized and counted on a hemocytometer. (B) Result of 5-bromodeoxyuridine (Brdu) incorporation with the treatments indicated in (A). BrdU-positive cells are presented as a percentage of BrdU-positive cells in the total of nuclear stained cells. The results are expressed as the means ± SEMs of 3 experiments. Different lowercase letters indicate statistically significant differences, P < 0.01.

Oral treatment of live EGF-LL stimulated the growth of early-weaned mice
The biological activities of EGF-LL were further tested in vivo by orally gavaging the live bacteria to early-weaned mice. A dose of 300 µL EGF-LL was delivered as whole cell suspension, together with the medium they were cultured in, twice daily into the stomach of mice for 9 d. During the experimental period, no abnormal behavior or diarrhea was observed. No significant difference was observed in food intake among the groups tested. The onset of BW change started on day 4; by day 9, the overall BW change in mice fed with EGF-LL (132.6 ± 12.0 g) was significantly higher than that of control mice in the PBS (117.3 ± 7.0 g) or EV-LL (121.1 ± 8.3 g) groups, but was comparable with that in the positive control group, ie, mice fed with 50 µg/kg rhEGF daily (129.6 ± 11.1 g; Figure 3A). Mice fed with EGF-LL appeared generally larger than that of the mice fed with PBS (Figure 3B).


Figure 3
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  		FIGURE 3. Effects of epidermal growth factor–expressing Lactococcus lactis (EGF-LL) on mouse growth in vivo. (A) Comparison of mean body weight changes after treatment for 9 d with EGF-LL (n = 16), L. lactis transformed with the empty vector backbone (EV-LL; n = 13), recombinant human EGF (rhEGF) (n = 11), or phosphate-buffered saline (PBS; n = 10). Body weight is expressed as the percentage of initial body weight for each respective animal. Data are expressed as means ± SEMs of 3 repeated experiments. Different lowercase letters indicate statistically significant differences, P < 0.01. (B) Representative photograph of body size differences of mice receiving the different treatments on day 9. The ruler represents the relative scale in centimeters.

EGF-LL–stimulated intestinal development of early-weaned mice
The morphology of the intestines was examined by measuring both the villous height and crypt depth of duodenum, jejunum, and ileum. Villi in all 3 segments of the intestine obtained from mice fed with EGF-LL were longer than those from the EV-LL and PBS groups. The differences were confirmed by a quantization study. The mean villous height of the duodenum, jejunum, and ileum were highest in the EGF-LL group (P < 0.001; Figure 4A). Interestingly, although lower than the EGF-LL and the positive control rhEGF group, the mean villous height of the EV-LL group was significantly greater than that of the PBS group in the duodenum, jejunum, and ileum. In terms of crypt depth (Figure 4B), there was no significant difference between the PBS, EV-LL, and rhEGF treatment groups at the duodenum and jejunum, whereas EGF-LL treatment resulted in a significantly increased crypt depth at all 3 segments of the small intestine (P < 0.01).


Figure 4
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  		FIGURE 4. Epidermal growth factor–expressing Lactococcus lactis (EGF-LL) enhanced intestinal development in early-weaned mice. The figure shows mean villous height (A) and crypt depth (B) in different compartments of the small intestine after the treatments. Cross-sections of duodenum, jejunum, and ileum were paraffin-embedded, sectioned, and stained in hematoxylin and eosin. At least 45 complete villous-crypt junctions of each segment of the individual mouse were measured. Data represent the means ± SEMs for 50 mice. Different lowercase letters indicate statistically significant differences, P < 0.01. PBS, phosphate-buffered saline; rhEGF, recombinant human epidermal growth factor; EV-LL, L. lactis transformed with the empty vector backbone.

EGF-LL increased the proliferation of intestine crypt cells
One of the possible mechanisms whereby EGF-LL increases villous height and crypt depth of the intestine may be via its stimulation of the proliferation of intestinal cells. PCNA is an auxiliary protein of DNA polymerases {delta} and {epsilon}—enzymes required for DNA synthesis (23). It is highly expressed at the S-phase of the cell cycle and therefore has been used as an indicator of cell proliferation. To examine the enterocyte proliferation status after the respective treatments, immunohistochemical analyses were performed on the paraffin-embedded intestinal tissue sections by using antibody against PCNA. As shown in Figure 5, {approx}80% of the PCNA-positive cells localized in the crypt area (arrows), whereas some proliferating cells had migrated along the villi (arrowheads). These representative micrographs also showed that the density of PCNA-positive cells in the EGF-LL and rhEGF groups was greater than that in the PBS and EV-LL groups. Negative controls were assayed with the same procedures, but the use of PCNA antibody was omitted to indicate the specificity of PCNA staining.


Figure 5
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  		FIGURE 5. Epidermal growth factor–expressing Lactococcus lactis (LL-EGF) increased proliferating cell nuclear antigen (PCNA)–positive cells. Representative images of immunohistochemical detection of PCNA in the intestine of mice treated with LL-EGF, L. lactis transformed with the empty vector backbone (LL-EV), recombinant human EGF (rhEGF), and EGF for 9 d. Cells were counterstained by hematoxylin to capture basic intestinal architecture. The negative control section was treated the same as the rest of the section, except that the PCNA antibody was omitted from the assay. Images were taken at x100 magnification; the scale bar is equivalent to 190 µm. Arrows indicate PCNA-positive (brown) cells, whereas arrowheads indicate PCNA-negative (blue) cells in the microvilli. PBS, phosphate-buffered saline.

Recombinant EGF and live EGF-LL were detected in the mouse intestine
Western blot analyses were performed to confirm the presence of EGF in the intestinal mucosal contents. A human polyclonal antibody derived from rabbit and raised against human recombinant EGF that has 100% cross-reactivity with pig EGF and <0.01% cross-reactivity with mouse EGF was used in the assays. Because of its specificity, the presence of the EGF signal in the blot should therefore represent the pig EGF that was being delivered to the intestine by EGF-LL. As shown in Figure 6, the 6-kDa EGF bands were detected in proteins extracted from the intestinal content and mucosal plus tissues of the EGF-LL but not the EV-LL mouse group. The presence of the 37-kDa GAPDH bands confirmed the equal loading of the protein samples.


Figure 6
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  		FIGURE 6. Porcine epidermal growth factor (pEGF) was detected in the small intestinal contents and tissues of mice that received EGF-expressing Lactococcus lactis (LL-EGF). Mucosal and tissue protein was collected from the intestines of mice fed with LL-EGF and L. lactis transformed with the empty vector backbone (LL-EV). Proteins were analyzed by Western blot immunodetection. Expression of EGF protein was detected not only in the intestinal contents (A) but in the intestinal tissues (B) of mice treated with LL-EGF for 9 d. No EGF protein was detected in the intestinal contents or tissues of mice fed with LL-EV. Loading control was shown by stripping the membrane and reblotting with GAPDH antibodies (C). D, duodenum; J, jejunum; I, ileum.

To study whether EGF-LL indeed survived in the small intestine, the intestinal contents of mice in the EGF-LL group were homogenized and plated on nutrient-rich agar plates. The recovery of recombinant L. lactis was selected by using erythromycin selection. Representative agar plates containing recovered erythromycin-resistant colonies from intestinal contents are shown in Figure 7A. A similar recovery rate was obtained from contents isolated from mice in the EV-LL group, whereas no bacteria were recovered on plates containing content homogenates of PBS or the rhEGF groups (data not shown). To confirm that the recovered bacterial colonies were EGF-LL, PCR-colony screening was performed by using specific primers to amplify the region spanning the P170 promoter and the transcriptional terminator. All 10 random colonies recovered from respective segments of the intestinal contents of mice in the EGF-LL group resulted in the 453-bp products (Figure 7B). Colonies recovered from the EV-LL group resulted in 303-bp PCR products (expected size minus EGF coding sequence; data not shown).


Figure 7
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  		FIGURE 7. Epidermal growth factor–expressing Lactococcus lactis (LL-EGF) survived in the small intestine of mice. (A) Representative photograph showing single colonies recovered from intestinal contents collected from mice that received LL-EGF treatments for 9 d. Ten randomly picked colonies were then selected from each plate of intestinal contents collected from individual animals for polymerase chain reaction colony screening. (B) Representative agarose pictures depicting the size of polymerase chain reaction product for EGF-positive clones.


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DISCUSSION
 
The presence of EGF has been identified in several physiologic fluids that directly interact with the intestine, for example, breast milk, bile, and saliva (5–7). The EGF receptor is a 170-kDa protein with tyrosine kinase activity. It was reported to be primarily localized on the basolateral surfaces of the enterocytes (24, 25). However, using autoradiographic techniques, Kelly et al (26) showed that EGF receptors are present on both microvillar and basolateral membranes of enterocytes of newborn and early-weaned pigs. In addition, the EGF receptor was also shown to be present on both the luminal and basolateral surfaces of small intestinal epithelial cells (27). Although controversy exists regarding the location of the EGF receptor, EGF may exert its effects via receptors present on the luminal surface. Alternatively, the greater mucosal permeability of the immature gut may allow EGF to bind to the EGF receptor in the basolateral membrane to mediate its effects in the intestine as previously suggested (28). Furthermore, it has been suggested that EGF receptors are 10 times more prevalent than EGF itself, which suggests that an exogenous supplement of EGF may be taken up well and used by developing animals (29).

In the intestine, enterocytes migrate from the crypts to the villous tip, epithelial proliferation is then followed by new enterocytes arising from stem cells in the crypt region of the intestine to help with the tissue repair process (30). The role of EGF on intestinal homeostasis has been well documented. In an organ fetal gut culture system, it was shown that EGF signaling is important for both the proliferation and survival of cells within the epithelial cell layer (31). EGF plays an important role in facilitating intestinal cell proliferation and thus positively influences the microscopic architecture of the intestine (31, 32). Our finding that oral administration of EGF-LL to newly weaned mice enhances intestinal development and is consistent with previous reports regarding the possible roles of EGF. Because multipotent crypt stem cells are present and divide within the crypt base, giving rise to the epithelial lineages, greater crypt depth indicates better developmental potential and more active intestinal development. The latter is also confirmed by our finding that intestinal epithelial cell proliferation is significantly enhanced by EGF-LL. The increased villous height provides a larger absorptive surface area. All these improvements may have enhanced the overall digestive and absorptive functions and thus resulted in greater BW gain in the EGF-LL mice.

Intestinal microbiota are important for the maintenance and recovery of intestine health (33, 34). They not only interact with intestinal epithelial cells and protect them from pathogens via interference with pathogen adhesion and invasion (35), but also play a role in defending against enteric infections (36). LAB are generally considered as safe organisms. Many of them are natural inhabitants of the intestinal tract of animals and humans (37). LAB have also been shown to play a role in the inhibition of pathogens in the intestine (38, 39). In fact, LAB are widely used probiotic bacteria, and are included in many supplement foods and shown to be beneficial for human and animal health (40). For example, when LAB included as formula supplement, the number of diarrhea and duration was decreased, and the number of days with fever, clinic visits, child care absences, and antibiotic prescriptions was reduced compared with infants in the control group (41). In the early-weaned piglets, the concentration of enteric LAB is reduced which results in the pigs being more susceptible of pathogen infections (42). This suggests that providing newly weaned pigs with LAB may help to restore the balance in the intestinal tract. In vivo studies have shown that in 30 out of 31 trials, supplementation of LAB in diets fed to weanling pigs has induced a positive growth response (43). Therefore, L. lactis itself, the vehicle used for producing and delivering EGF, is already a probiotic that is beneficial to intestinal health. This seems to be true when comparing villous height between the EV-LL and PBS control groups, although no BW difference was observed between the 2 groups.

There are several advantages to producing and delivering recombinant protein using L. lactis. L. lactis is a gram-positive bacterium consisting of a single cellular membrane that is ideal for secreting the recombinant protein (44); L. lactis has been proven to be highly efficient at expressing and secreting recombinant proteins (45); the detection of EGF in both the supernatant fluid and L. lactis cell pellet indicated that the expression system used in the current study also enabled the L. lactis to express and secrete EGF. Moreover, the fact that the supernatant fluid of the L. Lactis culture stimulates fibroblast proliferation indicates that the EGF secreted by recombinant L. lactis is indeed functional. Although Western blot analyses showed no significant differences in concentrations of EGF proteins collected at each time point, repeated measurements of OD600 showed that the growth of EGF-LL peaked at 24 h. This suggested that prolonged incubation of the cultures would not increase the yield of the recombinant protein. This finding agrees with data published by Steidler et al (46), from which the authors suggested that the production of secreted mouse interleukin-2 occurred predominantly during the exponential growth phase in L. lactis. The decrease in OD600 values at later stages of fermentation might have been due to glucose starvation or bacterial death due to aging.

With the use of the commercially available recombinant human EGF as a standard, it was estimated that the concentration of EGF in the culture supernatant fluid in a 24-h culture is {approx}500 ng/mL. Because similar amounts of EGF are also present in the cell pellet from the same volume of culture, the actual amount of EGF delivered is {approx}600 ng/d with feeding of 300 µL twice daily, which is equivalent to {approx}60 µg/kg daily. This dose of the growth factor was comparable with that of effective doses of other growth factors showing a stimulating effect on intestinal growth (47). In our study, live EGF transgenic L. lactis was recovered from duodenum, jejunum, and ileum, which indicated that recombinant L. lactis survives the stomach acids and the intestinal environment. This finding is consistent with the reports showing survival of the genetically marked L. lactis strain in the GI tract of human volunteers (48) and in the intestine of mice given IL-10–expressing L. lactis (17). This is not surprising because the digestive tract offers a relatively nonhostile environment and supply of nutrients that are required for the bacteria to survive. In addition, Western blot analysis showed a detectable level of EGF in the EGF-LL group but not in the control group, in which L. lactis with vector backbone was fed. This suggests that the recombinant L. lactis was capable of delivering EGF to all compartments of the intestine. Future study should optimize a delivery strategy that is biologically safe to use in an animal production setting and food chain. To this end, a passive biological containment approach for IL-10–expressing L. lactis has been successfully shown (18).

In conclusion, the current study showed that EGF-LL enhanced intestinal development and animal growth in early-weaned mice. This finding lays the foundation for developing a strategy to circumvent the problem of digestive dysfunction and the associated reductions in growth gain in other early-weaned animals. The combination of growth factor delivery and a probiotic approach may also offer possibilities for formulating dietary supplements for children during their weaning transition stage.


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ACKNOWLEDGMENTS
 
We thank Cheryl Soulliere and Julia Zhu for technical assistance.

The authors' responsibilities were as follows—QCKC: implemented the study and field investigations, analyzed and interpreted the data, and drafted the manuscript; ZY: generated the EGF-expressing L. lactis and reviewed the manuscript; PWD: conducted the BrdU staining and reviewed the manuscript; DW: helped design the study; KD: contributed to the experimental design, helped with the data analysis, and reviewed and edited the manuscript; and JL: designed and coordinated the implementation of the study, helped analyze and interpret the data, and drafted the manuscript. No conflicts of interest were declared.


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Received for publication October 8, 2008. Accepted for publication December 22, 2008.




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