Iron content of ferritin modulates its uptake by intestinal epithelium: implications for co-transport of prions
© Sunkesula et al; licensee BioMed Central Ltd. 2010
Received: 1 April 2010
Accepted: 29 April 2010
Published: 29 April 2010
The spread of Chronic Wasting Disease (CWD) in the deer and elk population has caused serious public health concerns due to its potential to infect farm animals and humans. Like other prion disorders such a sporadic Creutzfeldt-Jakob-disease of humans and Mad Cow Disease of cattle, CWD is caused by PrP-scrapie (PrPSc), a β-sheet rich isoform of a normal cell surface glycoprotein, the prion protein (PrPC). Since PrPSc is sufficient to cause infection and neurotoxicity if ingested by a susceptible host, it is important to understand the mechanism by which it crosses the stringent epithelial cell barrier of the small intestine. Possible mechanisms include co-transport with ferritin in ingested food and uptake by dendritic cells. Since ferritin is ubiquitously expressed and shares considerable homology among species, co-transport of PrPSc with ferritin can result in cross-species spread with deleterious consequences. We have used a combination of in vitro and in vivo models of intestinal epithelial cell barrier to understand the role of ferritin in mediating PrPSc uptake and transport. In this report, we demonstrate that PrPSc and ferritin from CWD affected deer and elk brains and scrapie from sheep resist degradation by digestive enzymes, and are transcytosed across a tight monolayer of human epithelial cells with significant efficiency. Likewise, ferritin from hamster brains is taken up by mouse intestinal epithelial cells in vivo, indicating that uptake of ferritin is not limited by species differences as described for prions. More importantly, the iron content of ferritin determines its efficiency of uptake and transport by Caco-2 cells and mouse models, providing insight into the mechanism(s) of ferritin and PrPSc uptake by intestinal epithelial cells.
Prion disorders are a group of neurodegenerative conditions of humans and animals that are known to be transmitted through ingestion of prion contaminated material. This mode of transmission was described historically as 'Kuru', a neurodegenerative condition of humans acquired by ingesting prion disease affected human brain tissue [1, 2]. Later, the disease was transmitted to humans from diseased cows, referred to as variant Creutzfeldt-Jakob disease (vCJD) [3–5]. Despite convincing evidence of its transmission through contaminated food, the mechanism by which PrP-scrapie (PrPSc), the principal pathogenic and infectious agent responsible for all prion disorders crosses the stringent epithelial cell barrier has remained enigmatic. Transport through dendritic cells, M cells, and co-transport in association with ferritin has been reported, but a complete understanding of either pathway is lacking [6–8].
PrPSc is a β-sheet rich isoform of a normal cell surface glycoprotein, the prion protein (PrPC) that acquires certain biochemical characteristics such as insolubility in non-ionic detergents, tendency to aggregate, limited resistance to digestion by proteinase-K, and the ability to replicate . Most infectious prion disorders are acquired when PrPSc from an exogenous source gains access to the central nervous system and induces the conversion of host PrPC to its own conformation. A certain amount of homology between the incoming PrPSc and host PrPC is required for efficient conversion, explaining the protective influence of species barrier such as between rodents and humans . However, the possible transmission of sheep scrapie to cattle and onward transmission to humans indicates that non-homologous PrPSc can be carried by certain hosts, and in some cases, convert host PrPC to a novel PrPSc conformation, albeit very slowly [11–13]. In view of these facts, it is important to evaluate whether PrPSc from deer and elk population infected with CWD can cross the species barrier and create a carrier state in cattle sharing the same grazing ground, or through contaminated food products in humans [14–17].
Since the most likely source of natural infection with CWD and other prion disorders is through ingestion of PrPSc contaminated food, it is important to understand the mechanism by which PrPSc, a protein of 27-37 kDa survives the harsh digestive environment and crosses the stringent epithelial cell barrier while retaining its infectious nature. The resilience of PrPSc to digestive enzymes is shared with ferritin, an iron storage protein that is a common constituent of all foods. In a previous report we demonstrated that PrPSc forms a relatively stable complex with ferritin in prion disease affected brain homogenates, and the complex is transcytosed together across a monolayer of Caco-2 cells, an in vitro model of human epithelial cell barrier [7, 18, 19]. Since ferritin shares significant homology across species, PrPSc from distant species is likely to ride 'piggy back' on ferritin to cross the epithelial cell layer, raising the possibility that infectious PrPSc from distant species such as deer and elk could cross the intestinal epithelial barrier of cattle or humans, and create a carrier state [20, 21].
To evaluate this possibility, we have checked the transport of ferritin from different species across a tight monolayer of Caco-2 cells, and confirmed our observations in vivo in mouse models. We report that brain ferritin from three different cases of CWD and sheep scrapie resists degradation by digestive enzymes (DE) and is associated with PrPSc. Both ferritin and PrPSc from diseased CWD and sheep brains are transported across a monolayer of Caco-2 cells, and the iron content of ferritin determines its efficiency of uptake and transport by Caco-2 cells in vitro, and by mouse intestinal epithelial cells in vivo. These observations have significant implications for inter-species spread of CWD and other animal prion disorders.
Chemicals and antibodies
Anti-PrP antibody 3F4 was obtained from Signet Laboratories (Dedham, MA, USA), anti-ferritin antibody from Sigma (St. Louis, MO, USA), anti-zonula occludens-1 (ZO-1) from Zymed (San Francisco, CA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Goat anti-Rabbit FITC (fluorescein isothiocyanate) and TRITC (tetramethylrhodamine B isothiocyanate) labeled secondary antibodies were from Southern Biotechnology Associates (Birmingham, AL, USA). Sulfo-NHS-biotin and streptavidin-Texas Red were purchased from Pierce (Rockford, IL, USA). All cell culture supplies were obtained from Invitrogen (Carlsbad, CA, USA). Nuclear stain Hoechst 33342 was obtained from Molecular Probes (Eugene, OR, USA). Radiolabeled 59FeCl3 was purchased from Perkin Elmer (Boston, MA, USA). All other reagents including desferroxamine (DFO) were procured from Sigma.
Preparation of brain homogenates, treatment with DE and proteinase K and immunoprecipitation
A 10% homogenate of normal, CWD, sheep scrapie, or sporadic CJD (sCJD) brain tissue was prepared in phosphate buffered saline (PBS, pH 7.4) by sonication on ice. In vitro digestion with enzymes was carried out as described in a previous report . Briefly, 0.5 ml of homogenate was incubated with 200 U of salivary amylase for 15 min at 37°C. The pH of the mixture was adjusted to 2.0 with 5 M HCl followed by addition of 50 μl of pepsin (4095 U) and incubation for 1 h at 37°C. Subsequently, the pH was raised to 6.0 with 1 M sodium bicarbonate and 0.2 ml of pancreatin-bile extract was added (1.85 mg of pancreatin and 11 mg of bile extract/ml of 0.1 M NaHCO3). After raising the pH to 7.4 with 6N NaOH, 8.4 μl each of 2 M NaCl and KCl solutions were added and the mixture was rocked for additional 2 h at 37°C. Digestive enzymes were inactivated by the addition of PMSF and protease inhibitors and samples were stored at -70°C till further use.
For PK treatment, the homogenate was mixed with equal volume of 2× lysis buffer (20 mM Tris-HCl; pH 7.4, 100 mM NaCl, 10 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) and incubated with 50 μg/ml of proteinase K (PK) for 1 h at 37°C. The reaction was stopped by the addition of PMSF and protease inhibitor cocktail. For co-immunoprecipitation studies, untreated or PK- or DE-treated normal homogenate (NH) and sCJDH samples were immunoprecipitated with anti-ferritin antibody as described previously, and eluted proteins were subjected to Western blotting and probed with 8H4, a monoclonal antibody specific to PrP .
Transport of PrPSc across Caco-2 monolayers
Caco-2 cells were cultured as described in an earlier report . In short, cells were resuspended in DMEM at a density of 2 × 108 cells/ml and added to the apical chamber (AP) chamber of poly-carbonate filters (Trans-well; 12 or 24 mm diameter; 3 μm pore size; Costar, Cambridge, MA) coated with collagen. The filters were placed in a 12- or 6-well culture dish containing 0.6 or 1.2 ml of DMEM respectively. The medium was replaced every day until the development of confluent monolayers with tight junctions (10-14 days). The integrity of tight junctions was monitored by measuring trans-epithelial electrical resistance (TEER) across the monolayer with a millicell-ERS instrument (Millipore, Bedford, MA). Monolayers exhibiting a TEER of > 400 Ώ/cm2 were used for transport studies.
To evaluate ferritin and PrPSc transport, Caco-2 monolayers were washed with serum-free medium, and 20 μl of sample diluted in 1 ml of serum-free medium was added to the AP chamber. The inserts were placed in a 6-well dish containing 1.2 ml of serum-free medium. After an overnight incubation at 37°C, medium from AP and basolateral (BL) chambers were collected, clarified for cell debris, and proteins were precipitated with cold methanol. Precipitated protein were boiled in sample buffer, immunoblotted, and detected with specific antibodies as described below.
Radiolabeling of cellular ferritin with 59FeCl3 and transport
Radiolabeling of mouse neuroblastoma N2a cells was performed essentially as described previously . Briefly, N2a cells cultured to 80% confluence were incubated in serum free medium for 1 h followed by incubation with 59FeCl3-citrate complex (1 mM sodium citrate and 20-25 μCi of 59FeCl3 in serum free Opti-MEM) for 4 h at 37°C. Following the incubation, cells were washed with ice cold PBS and lysed in PBS or native lysis buffer (0.14 M NaCl, 0.1 M HEPES, pH 7.4, 1.5% Triton X-100 and 1 mM PMSF). The amount of radioactivity incorporated was determined in cell lysates by γ-counting.
All animal procedures were performed as per guidelines established by the Animal Resource Center, Case Western Reserve University, and were based on protocols approved by the IACUC committee. Mice were fed with free 59FeCl3 or 59FeCl3-labeled N2a homogenates as described in a previous report . In short, Four months old FVB/NJ mice (Jackson Laboratory) were fasted overnight with water ad libitum and equal counts (300,000) of either free 59FeCl3 mixed with N2a cell homogenate or 59FeCl3 labeled N2a homogenates in (PBS) were administered orally using an olive-tipped gavage needle. After a chase of 4 h, mice were euthanized and blood was collected in heparinized vials. Brain, liver, spleen, and upper gastro-intestinal tract were harvested, washed with PBS, and snap frozen on dry ice. The organs were weighed, and incorporated radioactivity was counted in a γ-counter. Proximal region (1-2 cm) of duodenum was homogenized in native lysis buffer and analyzed for 59Fe labeled ferritin after resolving on native gradient gel followed by autoradiography. Native gradient gel electrophoresis of duodenum homogenates or N2a cell lysates for 59Fe-ferritin analysis was done as described by Vyoral et al.  with modification as in previous reports [22, 23].
Uptake of biotinylated proteins by mouse intestinal epithelium
Proteins in brain homogenates were biotinylated by adding 1 mg/ml EZ-Link Sulfo-NHS-Biotin followed by an overnight incubation on a rocking platform at 4°C. Biotinylated proteins were concentrated using methanol and suspended in 1 ml of 10% normal brain homogenate. Porcine bile extract was added to the suspended homogenate (11 mg of bile extract/ml of 0.1 M NaHCO3, pH 7.4), and contents were sonicated on ice. This comprised the control sample (-DFO). To deplete the sample of iron, brain homogenate was supplemented with iron chelator DFO (30 μM) and rocked for 1 h at 4°C. Following extensive dialysis against PBS at 4°C to remove DFO-iron complexes, the homogenate was biotinylated and prepared as above, and labeled as +DFO sample. FVB/NJ mice starved overnight were fed 0.2 ml of -DFO or +DFO homogenates by gastric gavage as above. After a chase of 2 h, the mice were sacrificed, and segments of intestine were processed for cryosectioning in Tissue tek OCT compound (Sakura Finetek USA Inc; Torrance, California) and snap frozen in isopentane cooled in Liquid nitrogen. Sections were cut at 5-10 μm thickness and immunostained.
Sections of mouse intestine were permeabilized with cold acetone at -20°C for 1 min and fixed in methanol for 20 min at -20°C. After rinsing with PBS, sections were incubated for 30 min in blocking buffer (PBS containing 3% non immune goat serum) at room temperature. Subsequently, sections were incubated with Streptavidin-Texas red diluted 1:40 in blocking buffer for 40 min at 37°C in a humidified chamber followed by three quick rinses and incubation with polyclonal anti-ferritin (1:10) antibody followed by goat anti rabbit FITC-conjugated secondary antibodies (1:20) for 40 min each. The sections were rinsed in PBS and incubated with monoclonal anti-ZO-1 (1:10) followed by anti-mouse TRITC conjugated secondary antibody (1:20). Stained sections were incubated with Hoechst 33342 (1 μg/ml) for 5 min to detect nuclei and mounted in Gel-mount (Biomeda Corp., Foster City, CA). Sections were then imaged by a fluorescent microscope. For immunostaining M17 cells, sub-confluent cultures were exposed to 5 μl of control or iron depleted samples, incubated for the indicated time, and processed for immunostaining as described in previous reports .
Detection of Iron
Prussian blue reaction was performed to detect iron in brain homogenates as described by Smith et al. . In brief, homogenates spotted on a PVDF membrane were immersed in a mixture of acidified potassium ferro- and ferri-cyanide solution (7%) for 20 min followed by washing with deionized water. Blue color indicated reactive iron.
SDS-PAGE and Western blotting
Proteins were processed for SDS-PAGE and Western blotting as described previously [27, 28]. Proteins transferred to PVDF membranes were probed with anti-PrP 8H4 (1:3000) or anti-ferritin antibody (1:1000) followed by horseradish peroxidase conjugated secondary antibody (1:6000). Immunoreactive bands were visualized by ECL detection system (Amersham Biosciences Inc.).
CWD and sheep PrPSc resist in vitro digestion and are associated with ferritin
To evaluate whether PrPSc from CWD affected brains forms a complex with ferritin as reported for sCJD , control and DE treated homogenates from normal (NH), sCJD (CJDH), and CWD-4 sample were immunoprecipitated with anti-ferritin antibody and checked for co-immunoprecipitation of PrP by Western blotting (Figure 1B). Following an overnight incubation with anti-ferritin antibody, protein-antibody complexes were captured with protein-A beads and washed using stringent conditions. Antibody-protein complexes eluted from beads (bds) and unbound proteins in the supernatant (sup) were fractionated by SDS-PAGE, subjected to Western blotting, and probed for PrP with 8H4. A significant amount of PrP from control (DE-) and faster migrating PrPSc from DE treated (DE+) CJDH and CWD-4 samples co-immunoprecipitates with ferritin (Figure 1B, lanes 2, 6, 8, 10, and 12). As expected, no PrP is detected in DE+ NH samples (Figure 1B, lanes 3 and 4, *), and in the supernatant of DE+ CJDH and CWD-4 samples (Figure 1B, lanes 7 and 11). Full-length PrP migrating between 27-37 kDa is detected in the supernatant and bead fractions of untreated NH, CJDH, and CWD-4 samples, indicating co-immunoprecipitation of normal (PrPC) and uncleaved PrPSc with ferritin (Figure 1B, lanes 1-2, 5-6, and 9-10). No PrP forms were detected in any sample in the absence of anti-ferritin antibody (data not shown). Thus, as demonstrated for sCJD , PrPSc and ferritin form a complex in CWD brain homogenates.
DE treated CWD and sheep scrapie are transported across Caco-2 monolayers
Although ferritin iron from distant species is taken up efficiently by human intestinal epithelium [30, 31], receptors specific for the uptake of ferritin have not been described. Nevertheless, PrPSc-ferritin complex from sCJD brain homogenates is transported across a monolayer of Caco-2 cells , suggesting that a similar phenomenon may occur for sheep scrapie and CWD prions. To evaluate this possibility, equal aliquots of DE treated samples from scrapie sheep and two cases of CWD were added to the apical (AP) chamber of a tight monolayer of Caco-2 cells cultured on filter inserts (Figure 1C). Following an overnight incubation at 37°C, culture medium from the AP and basolateral (BL) chambers was harvested, and precipitated proteins were fractionated by SDS-PAGE and transferred to a PVDF membrane. Probing for PrP reveals significant transport of DE-resistant PrPSc from sheep, CWD-1, and CWD-2 homogenates from the AP to the BL chamber (Figure 1D, lanes 1-6). Reprobing of the membrane for ferritin shows similar transport of ferritin to the BL chamber in all three samples (Figure 1D, lanes 1-6). To rule out possible leakage of proteins from AP to the BL chamber, resistance across filters supporting Caco-2 monolayers was checked before and after completion of the experiment, and the monolayers were stained for ZO-1, a tight junction protein, to confirm the integrity of tight junctions throughout the experimental procedure (Figure 1E, panels 1 and 2).
Together, these results demonstrate that a significant amount of PrPSc from scrapie infected sheep and CWD affected deer escapes digestion and is associated with ferritin. More importantly, ferritin and PrPSc from both species are transported across a monolayer of human Caco-2 cells, indicating that the species barrier against animal prions or ferritin is not stringent enough to block transport across these cells.
Iron content of ferritin determines efficiency of transport across Caco-2 cells
Ferritin is a major iron storage protein ubiquitously present in food products prepared from plant and animal sources, raising the possibility that it could serve as a significant source of dietary iron. Recent reports describe uptake of ferritin through specific receptors on oligodendrocytes, circulating reticulocytes, activated T and B-cells, HeLa cells, and K562 cells [32, 33], implicating ferritin as an iron delivery protein. Although the presence of similar receptors on Caco-2 cells is not known, we demonstrated co-transport of PrPSc and ferritin from sCJD brain homogenates across Caco-2 cells, though the underlying mechanism and whether ferritin or PrPSc drives transport of the complex was not assessed.
Brain ferritin is internalized by neuroblastoma cells
To evaluate whether neuroblastoma cells show similar selectivity for iron rich ferritin uptake as Caco-2 cells, normal human brain homogenate was treated with DFO and PK as above. Subsequently, equal amounts of methanol precipitated proteins from -DFO and +DFO samples resuspended in PBS were added to M17 cells cultured on glass coverslips. After an incubation of 30 minutes at 37°C, cells were washed with PBS, fixed in paraformaldehyde, permeabilized, and immunostained with anti-ferritin antibody followed by FITC-conjugated secondary antibody. A representative image shows significantly more uptake of -DFO treated ferritin by M17 cells relative to +DFO treated sample (Figure 2D, panels 1 and 2).
Taken together, these results suggest that efficiency of ferritin uptake by Caco-2 and neuroblastoma cells is determined in part by its iron content. Subsequent experiments were focused on confirming these results in vivo in mouse models.
Ferritin iron is taken up by mouse intestine
To evaluate whether ferritin is taken up by mouse intestinal epithelial cells, advantage was taken of the fact that ferritin from mouse neuroblastoma cells (N2a) migrates slower than mouse duodenal epithelial cell ferritin when fractionated on a native gradient gel, allowing differentiation between exogenously introduced N2a cell ferritin and endogenous duodenal ferritin.
Iron depletion reduces efficiency of ferritin uptake by mouse intestinal epithelium
Our data indicate that brain ferritin from deer, elk, and sheep is transcytosed across a monolayer of human Caco-2 epithelial cells, representing an in vitro model of human intestinal epithelium. The efficiency of ferritin transport is modulated by its iron content, suggesting that ferritin serves as a biological source of iron. A similar selection for iron-rich ferritin is noted in vivo in mice administered iron depleted mouse brain homogenate. Since PrPSc and ferritin form a complex in diseased brain homogenates and resist degradation by digestive enzymes, these observations suggest that ferritin could serve as a mediator of PrPSc transport across intestinal epithelial cells regardless of homology between incoming PrPSc and host PrPC.
Previous attempts at understanding the mechanism of PrPSc transport across the intestinal epithelial barrier have uncovered several possible pathways such as migrating dendritic cells and intestinal M cells . The involvement of laminin receptor (LRP/LR) dependent endocytosis of PrPSc has also been described [37, 38]. LRP receptors have been identified on the intestinal brush border, and ingested PrPSc has been reported to co-localize with these receptors following ingestion. It is believed that majority of ingested PrPSc loses infectivity following digestion . However, our observations suggest that majority of PrPSc and ferritin in the ingested material resist degradation by digestive enzymes, and are endocytosed by intestinal epithelial cells. Ferritin by itself is resistant to degradation and has been proposed as a source of dietary iron [30, 31, 40]. Whether association of PrPSc with ferritin increases its resistance to proteases in unclear at this time, but raises the possibility of co-transport with ferritin across the intestinal epithelium.
Regarding the uptake of ferritin, conflicting reports suggest either direct uptake followed by release of associated iron somewhere along the endocytic pathway, or release of iron in the intestinal lumen before uptake by epithelial cells through the DMT1 pathway. In support of the first hypothesis, receptors specific for ferritin uptake have been described on oligodendrocytes , activated B and T-cells, K562 cells, reticulocytes, and certain other cell lines , suggesting that ferritin is a common source of iron. Our observations indicate that ferritin is taken up by intestinal epithelial cells and accumulates in intestinal crypt cells before further transport or breakdown. Although our data fall short of identifying a specific receptor or demonstrating co-transport of PrPSc with ferritin due to technical reasons, our previous observations  and co-immunoprecipitation studies in this report leave little doubt that PrPSc and ferritin form a complex, and are likely to be co-transported across the intestinal epithelium.
This possibility raises significant public health concerns, especially due to the possibility of transmitting CWD prions from infected deer and elk to cattle and to the human food chain. Although PrPSc does not replicate unless the host PrPC and incoming PrPSc share significant homology, transport with ferritin could establish a carrier state in non-homologous hosts, resulting in disease at a later time. On the other hand, the association of PrPSc with ferritin provides possible avenues to reduce exposure and infectivity by chelating available iron in the infected material. Such a treatment would have the combined effect of decreased ferritin uptake and increased degradation of PrPSc by digestive enzymes [this report, ]. Future research is necessary to understand the mechanism of PrPSc-ferritin uptake by the intestine and practical ways to reduce infectivity due to accidental ingestion of PrPSc.
Thanks are extended to Subhabrata Basu, Sushovita Mukherjee, Soumya Ghosh, and Yuehua Gao for participating in some of the experimental procedures. This study was supported by grants R21AG033423 and R01NS044209 to NS from the National Institutes of Health.
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