Nononcogenic restoration of the intestinal barrier by E. coli-delivered human EGF

Abstract

Although mucoactive proteins, such as epidermal growth factor (EGF), could improve clinical outcomes of intestinal ulcerative diseases, their gastrointestinal application is limited because of their proteolytic digestion or concerns about tumor promotion. In the present study, ATP-binding cassette (ABC) transporter-linked secretion of human EGF from probiotic Escherichia coli (EGF-EcN) was created to promote beneficial actions of the EGF receptor, which is notably attenuated in patients with intestinal ulcerative injuries. Preventive and postinjury treatment with EGF-EcN alleviated intestinal ulcers and other readouts of disease severity in murine intestinal ulcer models. EGF-EcN administration promoted the restitutive recovery of damaged epithelial layers, particularly via upward expansion of highly proliferating progenitor cells from the lower crypts. Along with the epithelial barrier benefit, EGF-EcN improved goblet cell-associated mucosal integrity, which controls the access of luminal microbiota to the underlying host tissues. Despite concern about the oncogenic action of EGF, EGF-EcN did not aggravate colitis-associated colon cancer; instead, it alleviated protumorigenic activities and improved barrier integrity in the lesions. All findings indicate that probiotic bacteria-based precision delivery of human EGF is a promising mucosal intervention against gastrointestinal ulcers and malignant distress through crypt-derived barrier restoration.

Keywords: Inflammatory bowel disease; Microbiology; growth factors.

Figure 1. Construction and application of EGF-EcN in experimental gut models.

(A) EGFR expression levels in the colon or ileum of patients with Crohn’s disease (CD) or ulcerative colitis (UC). Three data sets (Sleiman’s, Gene Expression Omnibus [GEO] ID GSE10616, n = 58; Vemeire’s, GEO ID GSE75214, n = 194; and Haberman’s GEO ID GSE57945, n = 322) were compared. Haberman’s samples were only from the ileum from patients with UC or ileocolonic CD (iCD). The asterisks in box-and-whisker plots (min to max) represent significant differences between 2 groups (*P < 0.05, **P < 0.01, and ***P < 0.001 using 2-tailed, unpaired Student’s t test). The box plot depicts the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. (B) The scheme for the bacterial secretion of LARD3-linked EGF protein through prtDEF. Genes for human mature EGF polypeptide linked to 1 fragment of LARD3 and prtDEF in pKOV vector were encoded in the bacterial chromosome by a crossing-over recombination at the OmpC region. Based on SWISS-MODEL (https://swissmodel.expasy.org/), expected binding modes in the bacterial membrane are depicted, based on SWISS-MODEL. (C) Colonization activity of E. coli OP50 and EGF-EcN in C. elegans intestine in the presence of mucoadherent enteropathogenic E. coli (EPEC). Gut colonization was assessed in wild-type (N2) C. elegans after 24 hours of preincubation with mCherry-labeled nonpathogenic E. coli (OP50 or EGF-EcN) in the presence of GFP-labeled EPEC (original magnification, ×100). The pictures are representative of 10 independent observations. (D) Stool samples from EGF-EcN–treated mice were collected at indicated times after gavage. Colonized bacteria levels were estimated based on PCR with EcN-specific primers for Muta5/6. (E) GFP-labeled EGF-EcN (1 × 10⁹ CFU/200 μL) in the gut after gavage. The isolated intestines were observed on the 4th and 29th days after gavage. The colonizing bacteria were visualized in the intestine via an in vivo imaging system.

Figure 2. Actions of EGF-EcN in DSS-induced acute colitis.

Schematic overview of the DSS-induced colitis model with EGF-EcN treatment. (A) Six-week-old female mice were pretreated twice with vehicle, EcN, or EGF-EcN over 7 days (n = 12–15). The mice were then exposed to 3% DSS for 5 days to induce colitis. (B) Mouse body weight was monitored for indicated times after DSS exposure. The asterisks in the graph represent significant differences from mass changes of the DSS treatment group at each time point (*P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test). (C) Changes in colon length were measured on the 10th day after DSS treatment (the left box) and quantitatively analyzed (the right graph). The asterisks in the box-and-whisker plot (min to max) represent significant differences between 2 groups (*P < 0.05; **P < 0.01 using 2-tailed, unpaired Student’s t test). (D and G) Representative hematoxylin and eosin staining in intestinal lesions demonstrated using microscopy. Original magnification, ×100 (D), 400 (G). Scale bars: 100 μm. (E–G) Histopathological scores (E), levels of ulcer area (F), and neutrophil infiltration (G, red arrows) were compared between the DSS and EcN or EGF-EcN treatments. Results are shown as mean ± SEM (E) or box-and-whisker plots (min to max) (F and G). Different letters represent a significant difference between groups in each parameter (P < 0.05 using 1-way ANOVA with the Newman-Keuls post hoc test). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.

Figure 3. EGF-EcN–mediated actions in EGFR signaling in DSS-induced colitis.

(A–C) Six-week-old female mice were pretreated twice with vehicle, EcN, or EGF-EcN over 7 days (n = 12–15). The mice were then exposed to 3% DSS for 5 days to induce colitis. (A) Insulted colons were analyzed by IHC using the phosphorylated EGFR (p-EGFR) antibody with hematoxylin counterstaining. A microscopy image at original magnification of ×200 is shown at left. The relative density of p-EGFR was measured by using the HistoQuest tissue analysis software (each histogram of events with increasing diaminobenzidine (DAB) levels, described in the Methods section in detail). A quantitative comparison is shown in the right graphs. Scale bar: 100 μm. Results are shown as a box-and-whisker plot (min to max), and different letters represent a significant difference between groups (P < 0.05 using 1-way ANOVA with the Newman-Keuls post hoc test). (B) Each group of mouse colon lysates was subjected to Western blot analysis. The blots are representative of 3 independent experiments. (C) Eight-week-old female C57BL/6 mice were infected with 1 × 10⁹ EcN or EGF-EcN via oral gavage twice at 3-day intervals, and mouse gut was isolated on the third day after the second inoculation. LARD-tagged EGF secreted from the EGF-EcN and activated EGFR were detected in the colonic mucosa. The white arrows indicate EGFR-Tyr1068 (green) in the mucosa, and the numbers in the upper left represent the relative levels of LARD-EGF or p-EGFR. Relative levels of p-EGFR in mucosa were compared in the box-and-whisker plot (min to max) (right), and the asterisks represent a significant difference between 2 groups (***P < 0.001 using 2-tailed, unpaired Student’s t test).

Figure 4. EGF-EcN–mediated cell proliferation in DSS-induced colitis.

(A and B) Six-week-old female mice were pretreated twice with vehicle, EcN, or EGF-EcN over 7 days (n = 12–15). The mice were then exposed to 3% DSS for 5 days to induce colitis. (A) Sox-9 was detected and counterstained with hematoxylin (original magnification, ×200). Scale bar: 100 μm. Each histogram represents events with increasing DAB levels. A quantitative comparison is shown in the right graph. The asterisks represent significant differences between the 2 groups (**P < 0.01; ***P < 0.001; not significant [ns] using 2-tailed, unpaired Student’s t test). (B) Mouse colon tissues were stained for BrdU incorporation, and the nuclei were counterstained with hematoxylin (original magnification, ×100; inset magnification, ×600). Scale bar: 100 μm. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.

Figure 5. Links of EGFR expressions to gut barrier integrity.

(A and B) The EGFR expressions were correlated with genes associated with gut barrier integrity (CLDN3, TJP1, MUC2, LGR5, and SOX9) in intestines of patients with IBD (Sleiman’s, GEO ID GSE10616, n = 58; Vemeire’s, GEO ID GSE75214, n = 194; and Haberman’s GEO ID GSE57945, n = 194). Analysis of Vemeire’s samples was performed with colonic mRNA (B). All values were based on Pearson’s correlation analysis (R, Pearson’s correlation coefficient). (C–E) Six-week-old female mice were pretreated twice with vehicle, EcN, or EGF-EcN over 7 days (n = 12–15). The mice were then exposed to 3% DSS for 5 days to induce colitis. Claudin-3 (C), TJP1 (D), and cleaved caspase-3 (E) were detected and counterstained with hematoxylin (original magnification, ×200; inset magnification, ×800). Scale bar: 100 μm. A quantitative comparison is shown in each right graph. The asterisks represent significant differences between 2 groups (*P < 0.05; **P < 0.01; ***P < 0.001; ns using 2-tailed, unpaired Student’s t test).

Figure 6. Effects of EGF-EcN on gut epithelial and mucosal barrier integrity in colonic ulcer.

Six-week-old female mice were pretreated twice with vehicle, EcN, or EGF-EcN over 7 days (n = 12–15). The mice were then exposed to 3% DSS for 5 days to induce colitis. (A) Gut epithelia were stained with DAPI (blue) and phalloidin-TRITC (red) to stain nucleic acid and F-actin (original magnification, ×400; inset magnification, x8000). Scale bar: 100 μm. White arrows indicate the spread of apical F-actin into the basement membrane. TRITC, tetramethylrhodamine. (B) Colons were isolated for mucin analysis. Secreted and intracellular mucins were stained with Alcian blue (blue colored, original magnification, ×400. Scale bar: 100 μm). Each histogram represents events with increasing Alcian blue levels. A quantitative comparison is shown in the lower graph. (C) Gram staining to determine the accessibility of the total gut bacteria to the epithelial barrier (original magnification, ×400. Scale bar: 100 μm). The asterisks represent significant differences between 2 groups (each lower graph in A, B, and C) (**P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test). (D) Intestinal permeability was evaluated at 3 hours after oral administration with FITC-conjugated dextran (150 mg/kg body weight). Results are shown as the box-and-whisker plot (min to max), and different letters represent significant differences between groups (P < 0.05 using 1-way ANOVA with the Newman-Keuls post hoc test). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.

Figure 7. Involvement of EGFR signaling in recombinant bacteria–mediated protection.

Eight-week-old female C57BL/6 mice (n = 8–15) were treated twice with 3% DSS after oral gavages with 1 × 10⁹ EcN or EGF-EcN. Mice were then injected (intraperitoneally) with EGFR inhibitor (1 mg AG1478/mouse, Selleckchem) twice at 2-day intervals before and after the last day of DSS exposure. (A) Schematic outline of EGFR inhibition in the EcN or EGF-EcN treatment and DSS-induced colitis model. (B) Expression of p-EGFR in mucosa (green with the white arrow) was quantified (left box-and-whisker plot, min to max, based on fluorescence microscopy observations, shown at right) (*P < 0.05; **P < 0.01; ***P < 0.001; ns using 2-tailed, unpaired Student’s t test). (C) Representative hematoxylin and eosin (H&E) staining of the intestinal lesions as demonstrated by microscopy (original magnification, ×200). Scale bar: 100 μm. (D) Colons were isolated for analysis of goblet cells and mucin production and were stained with Alcian blue (original magnification, ×400. Scale bar: 100 μm). Each histogram represents events at an increasing Alcian blue level. A quantitative comparison is shown in the right graph (*P < 0.05; **P < 0.01; ***P < 0.001; ns using 2-tailed, unpaired Student’s t test). (E) Gram staining (original magnification, ×400. Scale bar: 100 μm). The asterisks represent significant differences between 2 groups (the right graph) (**P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test).

Figure 8. Effects of EGF-EcN on NSAID-induced ulcerative injuries in the small intestine.

Ten-week-old male mice were pretreated twice with vehicle, EcN, and EGF-EcN over 7 days (n = 12). The mice were then treated with 30 mg/kg of indomethacin via gavage. (A) At 24 hours after indomethacin treatment, tissues were observed in “Swiss rolls” of small intestines and analyzed using a stereoscopic microscope. The white arrows indicate hemorrhages along the line of intestines. Inset magnification, x18. (B–D) At 24 hours after indomethacin treatment, tissues were assessed for H&E staining patterns (B, upper), pathological severity score (B, lower), neutrophil infiltration (C, the representative images and the quantitation in the right graph), and ulcer area (D). Results in B are shown as mean ± SEM with asterisks representing significant differences between 2 groups (*P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test). Scale bars: 100 μm. Different letters (C) represent significant differences between groups (P < 0.05 using 1-way ANOVA with the Newman-Keuls post hoc test). The asterisks in the box-and-whisker plot (min to max, D) represent significant differences between 2 groups (*P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.

Figure 9. EGF-EcN–mediated actions in cell proliferation in NSAID-induced ulcer.

Ten-week-old male mice were pretreated twice with vehicle, EcN, and EGF-EcN over 7 days (n = 12). The mice were then treated with 30 mg/kg of indomethacin via gavage. (A) Mouse small intestinal tissues were stained for BrdU incorporation while the nuclei were counterstained with hematoxylin (original magnification, ×200). Scale bar: 100 μm. The BrdU-positive cells per villus were counted and the counts compared (***P < 0.001 and ns using 2-tailed, unpaired Student’s t test). (B) The vertical intestinal villus was divided into the crypt part and the middle villus part. BrdU-positive cells in each part were counted per villus. Results are shown as the box-and-whisker plot (min to max), and different letters represent significant differences between groups (P < 0.05 using 1-way ANOVA with the Newman-Keuls post hoc test). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. (C) IHC staining using anti–p-EGFR antibody was assessed under the microscope (original magnification, ×200). Scale bar: 100 μm. (D) Mouse small intestinal tissue was stained to detect Sox-9 protein and counterstained with hematoxylin. Each histogram represents events at an increasing DAB level. Scale bar: 100 μm. A quantitative comparison is shown in the graphs (C and D, *P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test).

Figure 10. Actions of EGF-EcN in colitis-associated tumorigenesis.

Tumors were induced in C57BL/6 mice by using AOM/DSS. (A) Experimental procedure of AOM/DSS exposure along with EcN and EGF-EcN treatment (n = 10–20). (B) Stool samples from mice were collected on the final day of the 12-week exposure as indicated in the treatment regime (A). Colonized bacteria were estimated based on PCR with EcN-specific primers for Muta5/6. (C) Mouse body weight change was monitored at indicated times after AOM injection. The asterisks in the graph represent significant differences from mass changes in the AOM/DSS treatment group at each time point (*P < 0.05 using 2-tailed, unpaired Student’s t test). (D) Colon length was compared. Results of quantitative analyses of colon length (upper) and symptoms in the anus (lower) are demonstrated. (E) Luminal parts of the colorectum used to detect adenomatous polyps (left) and a magnified illustration of the distal rectum (right). (F and G) Tumor number (F) and tumor area (G) based on the observation presented in E. The asterisks in box-and-whisker plots (min to max) (D, F, and G) represent significant differences between 2 groups (*P < 0.05; **P < 0.01; ***P < 0.001; ns using 2-tailed, unpaired Student’s t test).

Figure 11. (A) Tumor size distribution based on histological observations (*P < 0.05 using 2-tailed, unpaired Student’s t test).

(B) H&E staining of tumor mass (original magnification, ×100) and the frequencies of high- and low-grade dysplasia (right graphs). Scale bar: 100 μm. (C and D) The extent of tissue fibrosis in tumor mass of the AOM/DSS–induced cancer model was determined by examining Masson’s trichrome stain (C, original magnification, ×100) or IHC for alpha–smooth muscle actin (α-SMA) (D, original magnification, ×100) results. Scale bar: 100 μm. Each histogram represents events with increasing collagen (C) or α-SMA (D) levels. A quantitative comparison is shown in the right graph (*P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test).

Figure 12. Effects of EGF-EcN on the cell growth signaling in mice with colitis-associated cancer.

Tumors were assessed in C57BL/6 mice using AOM/DSS along with EcN and EGF-EcN treatment as depicted in Figure 9A (n = 10–20). (A) The expression of active EGFR in colons of the AOM/DSS–induced cancer model was compared (original magnification, ×200). Scale bar: 100 μm. Relative density of p-EGFR was measured by using HistoQuest tissue analysis software. (B) The extent of cell proliferation in colons of the AOM/DSS–induced cancer model was determined by measuring the expression of Ki-67 in the tumor mass (original magnification, ×200). Scale bar: 100 μm. Blue arrows indicate high expressions in the cryptic parts. (C) Sox-9 expression in colons of the AOM/DSS–induced cancer model was determined (original magnification, ×200). Scale bar: 200 μm. Each histogram represents events at an increasing DAB level. A quantitative comparison is shown in the right graph (*P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test).

Figure 13. Barrier regulation by EGF-EcN in colitis-associated cancer.

Tumors were assessed in C57BL/6 mice using AOM/DSS along with EcN and EGF-EcN treatment (n = 10–20) (A-C, E, F). (A) Colons (tumor mass and normal parts) were isolated for analysis of goblet cells and mucin production and stained with Alcian blue (original magnification, ×100. Scale bar: 100 μm). (B) Gut epithelia (tumor mass and normal parts) were stained with DAPI (blue) and phalloidin-TRITC (red) to stain nucleic acid and F-actin (original magnification, ×400; inset magnification, x8000). Scale bar: 100 μm. (C) ZO-1 expression in colons of the AOM/DSS–induced cancer model was determined (original magnification, ×100). Scale bar: 100 μm. Each histogram represents events at an increasing DAB level. A quantitative comparison is shown in the right graph (A–C, *P < 0.05; **P < 0.01; ***P < 0.001 using 2-tailed, unpaired Student’s t test). (D) HCT-8 cells were pretreated with AG1478 (10 μM) for 1 hour and then incubated with EcN or EGF-EcN for 12 hours. Cellular RNA was analyzed using quantitative PCR (n = 3; **P < 0.01 using 2-tailed, unpaired Student’s t test). (E and F) Neutrophil infiltration (E) in the tumor mass and serum cytokine levels (F) in the AOM/DSS–induced murine tumor model (*P < 0.05; ***P < 0.001 using 2-tailed, unpaired Student’s t test).