Correspondence Hiroyuki Nakayama, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Fax: +0081 5841 8185; email: email@example.com
To determine the mechanism of bovine intestinal incorporation of the pathogen, and the pathogenesis of prion protein in the early stage, cows suckling and weaning were orally given a fusion protein of Aβ-EGFP. Aβ-EGFP was incorporated through the villous columnar epithelial cells and accumulated in crypt patches in the ileum of suckling cows. The sites of the uptake and accumulation of Aβ-EGFP are very close to the peripheral nervous system; however, such uptake of Aβ-EGFP was not observed in 6-month-old post-weaning cows. The present study, therefore, suggests that the weaning period is very important for the risk of transmission.
sodium dodecylsulfate–polyacrylamide gel electrophoresis
Transmissible spongiform encephalopathies (TSE) are a group of fatal neurodegenerative diseases characterized by the abundant accumulation of abnormal prion protein with β-sheet structures (PrPSc) (1). The most probable entry site of PrPSc is the intestinal tract. After entry, transmissible agents may pass through one or several biological barriers and finally reach the brain. The peroral route of entry is widely assumed to be the most important in the natural pathogenesis of bovine spongiform encephalopathy (BSE), including scrapie (2), variant Creutzfeldt–Jakob disease (vCJD) (3) and other TSE (4). The transmission has also been shown to be caused by dietary exposure to PrPSc-contaminated food; however, the process of uptake and internal dynamics of infectious agents from the intestines to the central nervous system (CNS) is little understood (5).
Experimental models of oral transmission of BSE agent in minks (6), mice (7), sheep (8, 9), and non-human primates (10, 11) as well as scrapie agent in hamsters (12, 13), and mice (14) have been established; however, no reports regarding bovine models of oral transmission of BSE have been published. In such mouse and hamster models of oral transmission, the incubation period was longer than that of most other peripheral inoculations, and not all animals inoculated orally developed the disease. Various explanations may account for the difficulties of peroral transmission. First, the proteinaceous inoculum may be inactivated by digestive fluids and, second, a very limited amount of infectious agent may be transported from the intestinal lumen into the tissues. In addition, experiments on oral BSE infection using a BSE agent are quite difficult because scrapie agent cannot, but BSE agent can, infect human beings.
PrPSc was shown to be accumulated around follicular dendritic cells (FDC) of gut-associated lymphoid tissue (GALT) and in tangible-body macrophages of lymphoid nodules (15). PrPSc-positive cells with the morphology of dendritic cells and macrophages were also scattered in the dome region of intestinal Peyer's patches (16). This suggests that M cells in the follicle-associated epithelium are the entry site of transmissible agents (17); however, no report on the entry sites from the gut lumen and the distribution of PrPSc in the early stage of infection has been published. In addition, detection of a transmission agent orally inoculated into the intestinal tract is very difficult, because of dilution of the inoculum with gastrointestinal contents.
Recently, there was a report dealing with the PrPSc transport across the intestinal mucosa in sheep which were surgically operated to form an intestinal loop (18) and inoculated with PrPSc into the lumen. The report showed that the transepithelial passage through the intact villous epithelium was more probable than that through M cells in Peyer's patches.
In addition, suckling cows are thought to have the highest risk for food-born PrPSc contamination by statistical analysis (19). During the suckling period, the intestinal epithelium easily transports proteins such as immunoglobulins and growth factors in milk. Therefore, the intestinal epithelium during the suckling period may have a certain mechanism for incorporating prion proteins.
It was also reported that amyloid protein other than PrPSc was orally transmitted and induced systemic amyloidoses (20–22).
In the present study, to elucidate the uptake mechanism of transmissible agents into bovine intestines, a fusion protein of β-amyloid and EGFP which had been massively produced was given orally to suckling cows, instead of BSE agent. The produced protein is rich in a β-sheet structure like the BSE agent and can be used as alternative, because experimental β-amyloid does not require higher biosafety levels.
MATERIALS AND METHODS
Three 2-week-old and one 6-month-old Holstein cows and an 11-week-old F1 cow were housed in the Animal Resource Center, Graduate School of Agriculture and Life Science, the University of Tokyo. All animals were treated according to the regulated procedures authorized by the Animal Experiment Committee of the School.
Preparation of fusion protein
Fusion protein (Aβ-EGFP) of bovine β-amyloid protein structure (Aβ, 42 amino acids) at the N-terminal and enhanced green fluorescent protein (EGFP, 238 amino acids) at the C-terminal were mass produced by the following procedure. The fusion gene of bovine Aβ and EGFP was amplified by PCR. PCR products were then cloned into a pROX1-vector and subsequently constructed as Aβ-EGFP-expressing plasmid pROX1-Aβ-EGFP. Escherichia coli BL21 (DE3) Rosetta II was transformed with pROX1-Aβ-EGFP. The pROX1-Aβ-EGFP-harboring E. coli were incubated in SB medium supplemented with ampicillin at 20 °C for 18 hr after 1 mM isopropyl thiogalactoside (IPTG) induction. Culture cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C, and sonicated in PBS for 5 min on ice three times. The precipitated fraction of cell lysate was collected by centrifugation at 12 000 × g for 10 min at 4 °C and the pellet was washed with PBS containing 4% Triton X-100 at 4 °C. Repeating the washing procedure three times, the resultant pellet fraction was stored at −20 °C. Finally, 140 mL purified bovine Aβ-EGFP was produced. The concentration of Aβ-EGFP was determined by comparing the intensity of the band of the protein on SDS-PAGE with that of a known quantity of BSA (Sigma, St Louis, MO, USA). The final amount of 115 mg/mL Aβ-EGFP (38.1 g) was obtained.
Secondary structure analysis
Attenuated total reflection (ATR) FT-IR spectra (600–4000/cm) were measured using an FT-IR spectrophotometer (Nicolet 6700; Thermo Fisher Scientific K.K., Waltham, MA, USA) with ATR equipment containing a ZnSe ATR crystal, an ETC-Ever-Glo IR source, a deuterated lanthanum triglycine sulfate (DLATGS) detector, and a KBr beam-splitter under purging with a continuous flow of dried air (Air Tech Japan, Tokyo, Japan). The α-helix, β-sheet, β-turn and random coil contents in bovine Aβ-EGFP were estimated from the amide-I region of ATR IR spectra with ATR correction. Briefly, peaks of the amide-I region were first treated by Fourier self-deconvolution and then curve fitted using the Gauss and Lorentz formula with OMNIC Peak Resolve software (Thermo Fischer Scientific). The area corresponding to each secondary structure was calculated accordingly (23) and expressed as a percentage of the sum of areas measured by X-ray structure analysis (24).
Administration of Indian ink and Aβ-EGFP, and preparation of tissue specimens
First, an 11-week-old suckling cow (F1) was orally given 500 mL of 20% Indian ink (Kaimei, Saitama, Japan) diluted with PBS using a handmade intragastric feeding tube. Twelve hours after inoculation, the animal was anesthetized with propofol and killed by bleeding. The rumen, reticulum, omasum, abomasum, jejunum, ileum, colon, liver, and spleen were collected and fixed by immersion in 10% buffered formalin and embedded in paraffin by a conventional procedure. Paraffin sections (4 μm thick) were observed under a microscope.
Next, 2-week-old suckling cows (n = 3) and a 6-month-old cow (n = 1) were orally given 300 mL Aβ-EGFP diluted with PBS, pH 7.4 (10 mg/mL) or PBS alone (2-week-old cow; n = 1) using the same feeding tube. The same administrations were performed 12 and 15 hours after the first administration. Animals were then anesthetized with propofol and killed by bleeding 3 hours after the last administration. The duodenum, jejunum, ileum, colon, liver, and spleen were collected and fixed by immersion in 4% paraformaldehyde for 6 hr, and washed in PBS containing 6.8% sucrose. After dehydration in acetone for 1 hr, tissue samples were embedded in resin (Technovit 8100; Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer's instructions. Sections (4-μm thick) were sliced, mounted on silanized slide glasses, subjected to immunohistochemistry and observed under a fluorescent microscope (Olympus, Tokyo, Japan). The number of cells possessing Aβ-EGFP-specific fluorescence of villous epithelium in one microscope-visual field was counted. The number was counted at five random points. Statistical analysis between groups (P < 0.01) was carried out by Student's t-test.
Immunohistochemistry and lectin histochemistry
Aβ-EGFP in the tissues was confirmed by immunohistochemistry. Sections were pretreated with 0.01% trypsin at 37 °C for 10 min and then the non-specific reaction was quenched by the treatment with 0.3% hydrogen peroxide in methanol for 30 min. After incubating with anti-living colors full-length (EGFP) polyclonal antibody (1:200; Clontech, Mountain View, CA, USA) at 37 °C for 2 hr and with secondary horseradish peroxidase-coupled goat anti-rabbit immunoglobulin (Ig)G antibody (4 μg/mL; Nichirei, Tokyo, Japan) at room temperature for 30 min, the reaction resultants were colored with diamino benzidine (DAB; Wako, Osaka, Japan) was applied for 15 min. Sections were then counterstained with hematoxylin.
Resin sections were pretreated with 0.01% trypsin at 37 °C for 10 min. After incubation with anti-villin polyclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 2 hr and then with Alexa Fluor 546-coupled donkey anti-goat IgG antibody (5 μg/mL; Molecular Probes, Eugene, OR, USA) at room temperature for 30 min, sections were counterstained with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI). Villin is a marker for columnar epithelial cells with microvilli.
To observe the distribution in the peripheral nervous system and cellular prion protein in the bovine ileum, sections were reacted with anti-PgP 9.5 polyclonal antibody (1:100; Abcam, Tokyo, Japan) for 2 hr at 37 °C and with goat anti-rabbit IgG (H+L) antibody-coupled with Alexa Fluor 546 (5 μg/mL; Molecular Probes) at room temperature for 30 min. Other sections were reacted with anti-prion protein monoclonal antibody (T2; 1:100) or anti-PgP 9.5 polyclonal antibody (1:100; Abcam) for 2 hr at 37 °C, and further with Alexa Fluor 488-coupled goat anti-mouse IgG (H+L) antibody or goat anti-rabbit IgG (H+L) antibody-coupled with Alexa Fluor 546 (5 μg/mL; Molecular Probes) at room temperature for 30 min, respectively. Sections were then counterstained with DAPI.
Secondary structure analysis of produced Aβ-EGFP
Repeated massive incubation of E. coli transfected with the fusion gene produced 38.1 g Aβ-EGFP with 85% purity. The amide-I region of ATR FT-IR spectra of Aβ-EGFP was separated into nine peaks by peak resolution analysis based on the Gauss and Lorentz formula. These peaks revealed that the secondary structure of the Aβ-EGFP product consisted of 0%α-helix, 56.3%β-sheet, 33.5%β-turn, and 10.2% random coil structures (Fig. 1).
Uptake of carbon black through the ileal villous epithelium
Carbon black microparticles were incorporated into the villous epithelium of the ileum (Fig. 2e). The particles were attached to the surfaces of other gastrointestinal mucosa including the rumen, reticulum, omasum, abomasum, jejunum (Fig. 2a) and colon, although incorporation into the epithelium was not observed.
Uptake of Aβ-EGFP through the ileal epithelium
Aβ-EGFP was detected in the villous epithelium (Fig. 2f,j) and crypt patches (Fig. 2i) of the ileum of 2-week-old cows
Aβ-EGFP was incorporated into the cytoplasm but was not observed in the intercellular spaces (Fig. 3c). Such uptake of Aβ-EGFP was not observed in other intestinal regions (Fig. 2b). Although Aβ-EGFP were in the form of insoluble aggregated microparticles before administration, most of them diffusively existed in the intestinal lumen. This may indicate a certain digestion of the particles by gastric juice. The Aβ-EGFP deposition was also confirmed by immunohistochemistry using a specific anti-EGFP antibody (Fig. 3a,b). The uptake of Aβ-EGFP through ileal epithelial cells was also observed in a 6-month-old cow. The amount of incorporation into the epithelium was, however, much lower than in 2-week-old cows (Fig. 2l). Epithelial cells incorporating Aβ-EGFP were either villin positive or negative (Fig. 3c).
Distribution of peripheral nerve tissues and cellular prion protein in the intestinal wall
Peripheral nerve tissues were observed in the intestinal wall of a suckling cow, very close to the epithelial cells incorporating Aβ-EGFP and to the crypt patches possessing abundant Aβ-EGFP (Fig. 4a–c). In addition, such peripheral nerve cells contained a large amount of cellular prion proteins (Fig. 4d–f).
Secondary structural analysis by FT-IR revealed that the Aβ-EGFP used in the present study was an amyloidotic protein with a very abundant β-sheet structure. It has been reported that some orally given PrPSc was degraded in the stomach by gastric juice and digestive enzymes, and that PrPSc that escaped from the digestion can pass through the intestinal barrier (18). Aβ-EGFP may pass through the intestinal barrier in the same way. Subtle incorporation through the intestinal epithelial cells was detected by fluorescent imaging, but not by immunohistochemistry. Fluorescent imaging was, therefore, more sensitive than immunohistochemistry. Aβ-EGFP, which possesses abundant β-sheet structure, will be useful to analyze the mechanism of PrPSc uptake through the intestinal epithelium. To use infectious prion protein itself for such experiments is not appropriate because of its infectivity to human beings.
Villous epithelial cells are mainly composed of absorptive, goblet, endocrine, basal granular and Paneth cells (25). Villin is only found in microvilli of the digestive and urinary tracts. In the present study, Aβ-EGFP was mainly incorporated by villin-positive absorptive columnar epithelial cells in the ileum. A recent in vitro study (26) revealed that, in a case of coculture of intestinal epithelial cells and lymphoid cells, PrPSc was selectively incorporated into epithelial cells differentiated into M-like cells of the Peyer's patch, which play a role in incorporating foreign substances. In addition, the possible PrPSc uptake through the villous columnar epithelial cells in sheep was indicated recently (18), and the present study also revealed such incorporation of a β-sheet protein, Aβ, by columnar epithelial cells. In addition, suckling cows are thought to have the highest risk of BSE transmission (19), but Peyer's patches in suckling cows are still immature and M cells on Peyer's patches are very scarcely distributed in the ileum. Therefore, the uptake of β-sheet amyloid such as Aβ and PrPSc through villous columnar epithelial cells during the suckling period may be an important route for the intestinal entry of BSE agents. Importantly, during the suckling period, a protein with abundant β-sheet structures seems to be more easily incorporated through the ileal epithelium than that with less abundant β-sheet structures. Aβ-EGFP was incorporated during suckling periods in the mouse model, whereas albumin was degraded in the gastrointestinal tracts and only attached diffusively on the villous cell surface but was not incorporated (Ano et al., unpubl. data, 2008). In the present study, Aβ-EGFP was incorporated through ileal villous epithelial cells and accumulated in crypt patches also. These regions neighbor PgP 9.5-positive peripheral nerve tissues in the intestines which also express PrPc. In the case of oral or intragastric challenge with scrapie pathogen in hamsters, it is thought that infectious agents first accumulate in gut-associated lymphoid tissues or Peyer's patches and then move to the ganglia of the enteric nervous system (27). It has been thought that the distance between regions of PrPSc accumulation and the intestinal nerve tissues is very important for the entry of BSE agents to the nervous system (28). The results of the present study support this possibility. So, PrPSc incorporation through columnar epithelial cells may be the most plausible route of amyloid protein entry during the suckling period because Peyer's patches, which have been thought to be the route, are not yet developed before weaning.
In the present study, it was revealed that the uptake of amyloid proteins through the intestinal villous epithelium drastically changed at weaning. The uptake of Aβ-EGFP through the intestinal villous epithelium would be a superior model to study the incorporation of amyloid protein such as PrPSc into the body.
This work was supported by a Grant-in-Aid from the Food Safety Commission in the Japanese Government Cabinet Office.