. 2012 Dec;86(23):12731–12740. doi: 10.1128/JVI.01930-12

Rapid Transepithelial Transport of Prions following Inhalation

Anthony E Kincaid ^a,^b,^c,^✉, Kathryn F Hudson ^a,^*, Matthew W Richey ^a, Jason C Bartz ^c

PMCID: PMC3497678 PMID: 22973025

Abstract

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.

INTRODUCTION

The prion diseases are a group of fatal neurodegenerative diseases that affect animals, including humans. There is considerable evidence to support the hypothesis that the infectious agent is largely, if not entirely, PrP^Sc, a misfolded conformation of the normal prion protein, which is designated PrP^C (8, 12, 18, 51, 78, 85). Infectious prions are thought to be generated when PrP^Sc comes into direct contact with PrP^C and converts the native protein into a misfolded conformation. The misfolded isomer has distinct biological and physical properties, including the ability to self-propagate, aggregate, and cause disease (9, 14, 17, 24, 66).

While human prion diseases are rare, the prevalence of some animal prion diseases is relatively high and, in some cases, is increasing. For example, the prevalence of chronic wasting disease (CWD), a prion disease of deer and elk, can be as high as 15% in some free-ranging populations and 80 to 89% in captive herds (54, 55, 57, 87). The distribution of CWD in cervid populations in North America has expanded since the disease was first reported in Colorado and Wyoming in the 1980s, and with the recent identification of infected animals in Michigan, Texas, and Iowa, CWD has now been detected in 18 states and 2 Canadian provinces (15). One of the characteristic features of prion diseases is that they can be transmitted horizontally between animals, but neither the source nor the mode of transmission in free-ranging animal populations is known (56, 77). There is increasing evidence for the existence of infectious prions in a number of bodily fluids, including urine (31, 62, 74, 76), semen (70), saliva (31, 50, 53), nasal secretions (7), blood (13, 37, 53), milk (43, 47, 48), and feces (45, 71, 84). In addition, there is evidence that prion infectivity resides in the placenta (3, 68), decaying carcasses (56), antler velvet (4), and inanimate objects such as fence post and feeding trough surfaces (49). Infectious prions are particularly stable and have been shown to retain their infectivity after years in the environment; therefore, the ongoing transmission of disease in free-ranging animals is of great concern (10, 27, 40, 72, 75).

The oral route of infection has been established in the spread of prion diseases caused by the consumption of prion-contaminated food sources, including the bovine spongiform encephalopathy (BSE) outbreak in the United Kingdom (16), the spread of kuru in New Guinea (26), and the rare occurrence of transmissible mink encephalopathy (TME) that affected ranch-raised mink (52). While the oral route of infection may be involved in the transmission of disease in free-ranging animals, other routes of prion entry may exist (79). Recently the nasal cavity has been demonstrated experimentally to be an effective route of prion infection in hamsters, mice, and sheep following intranasal or extranasal (e.n.) inoculation of infected brain homogenate (6, 32, 42, 73). Furthermore, aerosolization of prions using a nebulizer, followed by exposure of the noses or snouts of mice, also resulted in the development of disease in cervidized, wild-type, and immunodeficient mice (19, 34). Given that animal populations that are susceptible to prion diseases use their highly developed olfactory systems for the detection of food and predators, as well as for reproductive and exploratory purposes, the mucosal surface of the nasal cavity may be a site of initial contact with inhaled materials harboring prions.

Whether the initial site of mucosal contact is in the gut or the nasal cavity, the infectious agent must be transported across a mucosal epithelium to gain entry into the body, where it can replicate and cause disease. There is disagreement about the location and mechanism of the transepithelial transport of prions following ingestion, with in vitro evidence for M cell transport (35), cotransport of prions with ferritin (58, 80), and laminin-mediated binding and endocytosis of prions (60), all across Caco-2 human epithelial cells. A second in vitro model of prion transport demonstrated that a mouse-adapted BSE agent crossed bovine M cells efficiently (59). In vivo experiments have demonstrated evidence for both M-cell-mediated and non-M-cell-mediated transepithelial transport of prion proteins following oral ingestion in mice and sheep, respectively (2, 20, 25, 36, 38, 46, 64, 83). The temporal parameters and the cell types mediating the transepithelial transport of prions within the nasal cavity have not been established.

The experiments reported here were designed to determine the location, cell type, and time course of the transepithelial transport of prions within the nasal cavity following inhalation by hamsters in an in vivo model of prion infection. An important component of these experiments was the ability to test the hypothesis that M cells located in the follicle-associated epithelium (FAE) that overlies the nasal mucosa-associated lymphoid tissue (NALT) are responsible for the transport of prions across the epithelium.

MATERIALS AND METHODS

Animal care.

Procedures involving animals were preapproved by the Creighton University Institutional Animal Care and Use Committee and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (63). Male adult golden Syrian hamsters from Harlan Sprague-Dawley (Indianapolis, IN) were group housed in standard cages with ad libitum access to food and water.

Animal inoculations.

Extranasal inoculations were performed as described previously (42). Hamsters receiving e.n. inoculations were first briefly anesthetized with isoflurane (Webster Veterinary, Kansas City, MO) and then placed in a supine position, and 10 to 100 μl of brain homogenate was placed just below each nostril (total volume, 20 to 200 μl). The brain homogenate was immediately inhaled into the nasal cavity; the animals began to move freely in their cages within 1 to 2 min of the start of the procedure.

Brain homogenate and control solutions.

Dulbecco's phosphate-buffered saline (Dulbecco's PBS), India ink (colloidal carbon in water), or a 10% (wt/vol) brain homogenate from either an HY TME-infected hamster (containing a 10^9.3 intracerebral 50% lethal dose [LD₅₀]/g) or an uninfected hamster was used. India ink was used to identify M cells in the FAE of the hamster nasal cavity (69).

Tissue collection.

Animals exposed to PBS, India ink, or infected or mock-infected brain homogenate were reanesthetized following a specified survival period and were transcardially perfused with 50 ml of 0.01 M Dulbecco's PBS, followed by 50 to 75 ml of McLean's paraformaldehyde-lysine-periodate (PLP) fixative. Skulls were removed and were placed in PLP at room temperature overnight; then they were decalcified for 2 weeks at room temperature with a solution change at 1 week (decalcifying solution; Richard-Allan Scientific, Kalamazoo, MI). By using the hard palate for orientation, the nasal cavity was excised between the posterior edge of the incisors and the anterior edge of the seventh palatal ridge, a length of about 12 mm. This block of tissue contains the entire nasal cavity, including the NALT and the olfactory and respiratory epithelia, but not the rostral vestibule, which is lined by a stratified squamous epithelium (1). This block was then cut and divided into three 4-mm coronal slices, or sagittally into single right and left halves, which were placed in a cassette for paraffin processing and embedding. Serial sections (7 μm) were cut with a rotary microtome and were mounted onto glass slides. A small number of animals (2 untreated and 2 PBS-inoculated animals) were not transcardially perfused but were immersion fixed in PLP for 1 week prior to decalcification and paraffin processing in order to differentiate lymphatic vessels from blood vessels.

Histology.

To establish the locations and boundaries of the various types of epithelia found within the nasal cavity, including the location of M cells, tissue sections from untreated animals or from animals extranasally exposed to India ink or PBS were cleared, dehydrated, and stained with periodic acid-Schiff stain (PAS) or hematoxylin and eosin (H&E). Pairs of tissue sections not further than 112 μm apart through the rostral-caudal extent of the hamster nasal cavity were examined using an Olympus BX 40 light microscope to demonstrate normal nasal mucosal morphology and the location of M cells.

IHC.

Immunohistochemistry (IHC) was performed to detect PrP^d as reported previously, and infected and mock-infected tissues were processed at the same time using the same reagents (42). In brief, tissue sections were deparaffinized and were subjected to antigen retrieval in formic acid (95%) for 10 min at room temperature. All subsequent steps were carried out at room temperature; incubations were separated by 3 rinses with 0.05% (vol/vol) Tween in Tris-buffered saline (TTBS). Endogenous peroxidase and nonspecific antibody binding were inhibited by incubating the tissue sections in 0.3% H₂O₂-methanol for 20 min, followed by incubation in 10% normal horse serum in TTBS for 30 min. The prion protein was visualized by using the avidin-biotin method. Incubation in an anti-prion protein monoclonal antibody (clone 3F4; dilution, 1:600; Chemicon, Temecula, CA) was carried out for 2 h at room temperature, followed by incubation in a biotinylated horse anti-mouse antibody (dilution, 1:600; Vector Laboratories, Burlingame, CA) for 30 min. The sections were placed in ABC solution (dilution, 1:200; Vector Laboratories, Burlingame, CA) for 15 to 20 min and were subsequently reacted in a solution containing filtered 0.05% diaminobenzidine tetrachloride (Sigma, St. Louis, MO) with 0.0015% H₂O₂. The sections were counterstained with hematoxylin, dehydrated through graded alcohols, and placed on coverslips with Cytoseal-XYL (Richard-Allan Scientific, Kalamazoo, MI). Glial fibrillary acidic protein (GFAP) IHC was carried out in a similar manner with the following differences: there was no antigen retrieval step; the blocking step was carried out using normal goat serum; the primary antibody was a rabbit anti-GFAP polyclonal antibody (dilution, 1:800; Dako, Carpinteria, CA); and the secondary antibody was a biotinylated goat anti-rabbit antibody (dilution, 1:800, Vector Laboratories, Burlingame, CA). Some sections were processed identically with either the primary or secondary antibodies omitted, or with a mouse immunoglobulin G isotype control (Abcam, Cambridge, MA) in place of the primary antibody at the same concentration. Pairs of tissue sections not further than 56 μm apart were examined using an Olympus BX 40 light microscope. When additional morphological detail was required, adjacent tissue sections were processed for GFAP IHC or were stained with H&E.

RESULTS

Hamsters are obligate nose breathers, so a small amount of brain homogenate placed just below their nostrils was immediately inhaled into their nasal cavities. This behavior precluded the need for placing a pipette inside the nose, where it could injure the nasal mucosa and expose brain homogenate directly to blood vessels and nerves. Mucosal damage could lead to direct hematogenous spread and/or neuroinvasion, which would complicate the interpretation of the transepithelial transport route and mechanism(s).

Following inhalation of PBS (n = 7), India ink (n = 6), and infected (n = 35) or uninfected (n = 15) brain homogenate, hamsters were allowed to survive for 1 to 180 min prior to the initiation of transcardial perfusion. Control animals were left untreated (n = 6). Regularly spaced tissue sections were inspected for the presence of ink, GFAP, or PrP^d. The average number of immunohistochemically processed tissue sections analyzed per animal was 366 (range, 150 to 640). Sagittally sectioned nasal cavities produced fewer tissue sections, because the nasal cavity is approximately twice as long as it is wide. GFAP is the principal intermediate filament of astrocytes and therefore is a component of both infected and uninfected brain homogenates, while PrP^d is found only in prion-infected brain homogenate (8, 14, 21). Roughly two-thirds of the tissue sections were processed using GFAP IHC, since tissue morphology is better preserved by this procedure than by PrP^d IHC, which requires the use of formic acid for antigen retrieval. The mucosal surface and underlying lamina propria of tissue sections through the rostral-caudal extent of each nasal cavity were inspected using a light microscope, and brain homogenate was identified with either GFAP or PrP^d IHC. Brain homogenate was identified in the nasal cavity of each experimental animal but was not seen in untreated animals or in those who inhaled PBS. While more brain homogenate was observed within the nasal cavities of animals that inhaled larger volumes of brain homogenate, the volume of brain homogenate was not related to the occurrence of transport. Therefore, the data for animals with the same survival time that had inhaled different volumes of brain homogenate were combined. Photomicrographs were taken wherever brain homogenate was identified on the surface of, within, or beneath the nasal mucosa. Omission of either the primary or the secondary antibody or replacement of the primary antibody with an isotype control resulted in a complete lack of staining (data not shown).

Hamster nasal cavity and light microscopic identification of M cells.

The hamster nasal cavity is lined by a variety of morphologically distinct epithelia, including the respiratory and olfactory epithelia and the FAE, which lies adjacent to the NALT (Fig. 1A and B). M cells are nonciliated with a flattened apical surface and distinct intraepithelial pockets that contain lymphoid cells (28, 44) and were identified in the FAE overlying the NALT in H&E- and PAS-stained tissue sections from untreated animals (Fig. 1C). M cells could also be identified based on their ability to endocytose inhaled India ink (69). Ink particles were identified within M cells in all 6 animals that were allowed to survive for 5 to 30 min after inhalation of 20 to 100 μl of ink (Fig. 1D). Inspection of untreated or PBS-exposed animals did not reveal any visible particulate matter on, or within, the M cells of the FAE of the nasal cavity (Fig. 1C). The average number of tissue sections per animal that were processed for H&E and PAS staining was 178 (range, 136 to 204).

Fig 1 — Hamster nasal cavity epithelial types and the identification of M cells. (A) Low-power view of a PAS-stained portion of a hamster nasal cavity demonstrating the morphology of the olfactory and respiratory epithelia (OE and RE, respectively) (box 1, enlarged in panel B) and the FAE (indicated by asterisks) with M cells overlying the NALT (box 2, enlarged in panel C). (B) The RE can be distinguished from the OE based on the thickness of the epithelial layer and the length of cilia. (C) M cells can be identified by their distinct morphology, including intraepithelial pockets (outlined) and the lack of cilia, which distinguish them from neighboring goblet and respiratory epithelial cells. (D) An H&E-stained tissue section from a hamster extranasally exposed to 20 μl India ink that survived for 30 min prior to perfusion. Note the presence of ink particles within a subset of the M cells of the FAE. Asterisks indicate air space. Bars, 200 μm (A) and 20 μm (B, C, and D).

Identification of inhaled prions.

Prion-infected or uninfected brain homogenate was identified within the nasal cavity of each animal following the inhalation of brain homogenate from either HY TME-infected or mock-infected animals. By use of GFAP IHC, prion-infected or uninfected brain homogenate was easily identified as aggregated or punctate brown particulate matter within the nasal cavity airspace and lining the nasal mucosa (Fig. 2). By use of PrP^d IHC, prion-infected brain homogenate was identified as small brown punctate particulate matter, while uninfected brain homogenate was unstained (Fig. 2). PrP^d IHC was used on tissue sections adjacent to GFAP-positive sections to confirm the presence of PrP^d in brain homogenate in animals that had inhaled infected brain homogenate. Brain homogenate from HY TME-infected animals was immunopositive for GFAP and PrP^d, while brain homogenate from uninfected animals was immunopositive for GFAP but not for PrP^d (Fig. 2).

Fig 2 — Uninfected and infected brain homogenates in the hamster nasal cavity after inhalation, identified by GFAP IHC and PrP^Sc IHC. (A) Low-power view of a portion of a nasal turbinate in the nasal cavity of a hamster that had been inoculated extranasally with mock-infected brain homogenate and that survived for 5 min prior to perfusion, processed using GFAP IHC. The boxed area is enlarged in panel B. (B) Higher-power view of the uninfected brain homogenate identified using GFAP IHC. (C) Higher-power view of an adjacent tissue section processed using PrP^Sc IHC. Note the lack of punctate immunoreactivity, indicating that the brain homogenate contains GFAP but lacks PrP^Sc. (D) Low-power view of a portion of a nasal turbinate in the nasal cavity of a hamster that had been inoculated extranasally with HY TME-infected brain homogenate and that survived for 5 min prior to perfusion, processed using GFAP IHC. The boxed area is enlarged in panel E. Note the GFAP-labeled bundle of nonmyelinating Schwann cells in the lamina propria (indicated by an arrow). (E) GFAP-labeled brain homogenate from a HY TME-infected animal. (F) An adjacent tissue section processed using PrP^Sc IHC. Note the distinct punctate labeled brain homogenate that is characteristic of PrP^Sc, indicating that this section contains GFAP and PrP^Sc. Contrast the staining in panels C and F to note the distinction between PrP^Sc-negative and PrP^Sc-positive brain homogenates. Asterisks indicate air space. Bars, 100 μm (A and D) and 20 μm (B, C, E, and F).

M cell transport of brain homogenate.

Based on previous reports of M cell involvement in the transport of prions, M cells in the FAE adjacent to the NALT were examined using a light microscope. Infected or mock-infected brain homogenate was identified using GFAP IHC and was seen adhering to the FAE of 44 of 50 animals following extranasal inoculation (Table 1; Fig. 3). The criterion used for positive identification of brain homogenate or prions within M cells of the FAE was brown punctate GFAP or PrP^d immunoreactivity surrounded by M cell cytoplasm in the same focal plane. By applying this criterion, brain homogenate, either infected or uninfected, was identified within M cells in 31 of the 43 hamsters that survived for 5 min or longer (Table 1; Fig. 3). The number of M cells noted to contain brain homogenate for each animal at each time point was counted to determine the period of maximal M cell transport of brain homogenate. The greatest number of M cells containing brain homogenate was found in those animals that survived 10 to 60 min prior to the initiation of perfusion, with a noticeable drop in the number of M cells containing brain homogenate in those animals that survived 180 min (Table 1). Analysis of tissue deep within the M cell layer was restricted to tissue sections processed for PrP^d IHC, since GFAP labels thin-diameter nonmyelinating Schwann cells (29, 39), which are located deep within M cells, in the NALT, and coursing through the lamina propria (Fig. 2D and 4E).

Table 1.

Brain homogenate distribution in the nasal cavity following inhalation

Survival time^a (no. of animals)	Inoculum (vol [μl])^b	No. of animals with brain homogenate at the indicated location/total no.
Survival time^a (no. of animals)	Inoculum (vol [μl])^b	On FAE	Inside M cells (no. of M cells)	Crossing between epithelial cells	Deep within epithelium
1–2 min (7)	Uninfected bh (50–100)	2/2	0/2	2/2	2/2
1–2 min (7)	Infected bh (50–200)	5/5	0/5	4/5	5/5
5 min (16)	Uninfected bh (20–100)	6/6	2/6 (5)	5/6	6/6
5 min (16)	Infected bh (20–200)	10/10	8/10 (15)	9/10	9/10
10–20 min (13)	Uninfected bh (100)	2/3	2/3 (7)	1/3	2/3
10–20 min (13)	Infected bh (100)	10/10	10/10 (44)	10/10	10/10
60 min (7)	Uninfected bh (100)	1/2	1/2 (2)	1/2	2/2
60 min (7)	Infected bh (50–100)	5/5	5/5 (29)	5/5	5/5
180 min (7)	Uninfected bh (100)	1/2	1/2 (3)	1/2	2/2
180 min (7)	Infected bh (100)	2/5	2/5 (4)	2/5	4/5

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Time to the initiation of perfusion. The time until death included an additional 10 to 12 min.

Total volume of mock-infected or HY TME-infected brain homogenate applied below the nostrils. bh, brain homogenate.

Fig 3 — Transcellular transport of prions by M cells in the nasal cavity following inhalation. (A and B) Inhaled mock-infected brain homogenate (A) and prion-infected brain homogenate (B) were identified with GFAP IHC located on the surface of the FAE of hamsters. Note the lack of brain homogenate within the M cells of the FAE in both of these sections: one taken from an animal that survived 5 min before perfusion (A) and the other from an animal that survived 1 min before perfusion (B). (C and D) Infected brain homogenate identified with GFAP IHC (C) and PrP^Sc IHC (D) within M cells (indicated by arrows) of animals that survived 10 min (C) or 5 min (D) before perfusion. Asterisks indicate air space. Bars, 50 μm (A and B) and 20 μm (C and D).

Fig 4 — Paracellular transport of brain homogenate in the nasal cavity following inhalation, identified with GFAP IHC. Shown are inhaled mock-infected (A) and infected (B) brain homogenates crossing the nasal mucosa between cells of the olfactory epithelium 5 min (A) and 1 min (B) after inhalation. Brain homogenate could be seen between epithelial cells spanning the complete width of the epithelium and entering the underlying lamina propria on some tissue sections (C). Note that multiple examples of paracellular transport of infected (B, C, and D) and uninfected (E) brain homogenates were observed on some tissue sections. Transport of brain homogenate was noted across the olfactory (C) and respiratory (D) epithelia and the FAE (E). Nonmyelinating Schwann cells deep within the FAE (indicated by arrows) are labeled with GFAP IHC. Note that the width of the paracellular transport appears to be approximately the same as that of a single epithelial cell in all panels. NS, nasal septum; OE, olfactory epithelium; RE, respiratory epithelium. Bars, 100 μm (A and B) and 25 μm (C, D, and E).

Paracellular transport of brain homogenate occurs between olfactory epithelium, respiratory epithelium, and FAE cells within minutes of extranasal inoculation.

Infected and uninfected brain homogenates were detected penetrating or completely spanning the nasal mucosae in 40 of the 50 animals inoculated extranasally in this experiment (Table 1; Fig. 4A and B). This was determined to be paracellular transport, because brain homogenate was located between intact epithelial cells, not within cells. This was confirmed by focusing through the depth of the tissue section and by examining the same cells on adjacent tissue sections. Paracellular transport of brain homogenate was identified between cells of the respiratory and olfactory epithelia and the FAE of the nasal cavity (Fig. 4) and could be seen extending into the underlying lamina propria in some cases (Fig. 4C and D). There were multiple examples of transepithelial transport in all animals exhibiting transport; in some cases, multiple examples of paracellular transport were identified on single tissue sections (Fig. 4B, D, and E). While paracellular transport was seen in animals from all survival time points, the number of animals demonstrating paracellular transport decreased by about 50% among those that survived 180 min following inoculation (Table 1). There were no examples of brain homogenate crossing the keratinized stratified squamous epithelium that lines the vestibule of the proximal nasal cavity (33).

Prions quickly enter lymphatic vessels in the lamina propria but not the NALT.

Infected or uninfected brain homogenate was identified within the lumens of vessels in the laminae propriae of 47 of the 50 inoculated animals in this study (Table 1). These vessels were determined to be predominantly lymphatic vessels and not blood vessels. This distinction was based on the analysis of vessels from immersion-fixed animals. Immersion fixation does not rinse the blood cells from blood vessels, so blood vessels were easily distinguished by the presence of red blood cells within their lumens, and lymphatic vessels by the lack of blood cells. The locations of lymphatic vessels were found to be relatively constant between animals, especially for medium- and large-diameter lymphatic vessels. Infected or uninfected brain homogenate was identified inside the lumens of lymphatic vessels in the laminae propriae of the nasal cavities of animals at all survival time points (Fig. 5). PrP^d was not identified in the intraepithelial pockets of M cells, adjacent to the NALT, or within the NALT for any animal in this study.

Fig 5 — Brain homogenate was located within the lumens of lymphatic vessels of the lamina propria of the nasal cavity within minutes of inhalation and was still visible for hours after inhalation. Infected (A and D) and uninfected (B and C) brain homogenates (indicated by arrows) were located within lymphatics 5 min (A and B) and 60 min (C and D) after inhalation. Bars, 50 μm.

DISCUSSION

The in vivo transport of infected brain homogenate containing PrP^d across the nasal mucosa began within minutes of inhalation, and transport could still be detected in some animals as late as 180 min following inhalation. Paracellular transport was detected in animals that were perfused within 1 to 2 min after inhalation, while M cell transport was first detected in animals that were perfused beginning 5 min after inhalation. Both types of transport were detected in all remaining survival groups, with a noticeable decrease in the number of animals demonstrating transport among those that survived 180 min after inhalation. The rapid and prolonged period of transepithelial transport of PrP^d in the nasal cavity following inhalation is consistent with the results of a study performed in a sheep gut-loop model of prion infection where prion proteins were detected in the lumens of villous and submucosal lymphatics 15 min to 210 min following direct inoculation of an isolated loop of distal ileum in sheep (38). The time frame, the type of transport (transcellular or paracellular), and the cells/epithelia involved in the in vivo transport of prions following inhalation have been identified. Most notably, the paracellular transport of relatively large amounts of brain homogenate between epithelial cells in the nasal cavity identified here has not been described previously. While the sequence of events leading to the paracellular transport of prions has not been determined, the mechanism of transepithelial transport may involve the passage of brain homogenate through existing gaps in the mucosa created by the natural shedding of epithelial cells that has been reported in the intestines of mice and humans (11, 41, 86). Utilization of existing gaps in the epithelia would be consistent with the observed speed and physical dimensions of the transport of brain homogenate reported here, where the passage of brain homogenate between cells often appeared to be about one epithelial cell in width and occurred within minutes of inhalation (Fig. 4).

The persistence and transepithelial transport of brain homogenate in the nasal cavity for as long as 3 h after inhalation, reported here, are surprising, since nasal mucociliary activity results in measured mucus flow rates ranging from 0.9 to 11 mm/min in different areas of the rat nasal cavity and to an average of 1.3 mm/min in the mouse nasal cavity (30, 61). At these rates, inhaled brain homogenate would be expected to be cleared from the hamster nasal cavity in less than 60 min. This prolonged period of transport is similar to that reported for the sheep gut-loop model, where prions were detected in submucosal lymphatics for as long as 3.5 h following inoculation (38).

A goal of these experiments was to test the hypothesis that prion entry following inhalation was mediated via M cells of the FAE in the nasal cavity. Infected or uninfected brain homogenate was seen within a relatively modest number of M cells (average, 1 to 5 per animal) in 31 of 50 animals in this study (Table 1). Given that the efficiency of this route of inoculation has been determined to be 10 to 100 times greater than that of per os inoculation (42), the number of animals demonstrating M cell transport and the number of M cells per animal transporting brain homogenate in this study were lower than might be expected. This is in marked contrast to the robust paracellular transport, reported here, of PrP^d in brain homogenate that was noted between cells of the olfactory, respiratory, and follicle-associated epithelia that line the nasal cavity (Table 1; Fig. 4). The results of this study are consistent with a relatively minor role for M cell transport in the transepithelial transport of prions following inhalation, with a greater contribution coming from the paracellular transport of prions. The limited number of examples of M cell involvement in PrP^d transport following inhalation was not due to a lack of exposure to brain homogenate, since almost all of the animals examined in this study had multiple examples of brain homogenate resting on FAE (Table 1). There are certain limitations to the interpretations that can be made about a dynamic cellular process that occurs across a surface and over a period when one is examining sections taken at specific time points. First, the transport of brain homogenate across the M cells could happen very quickly, so that the chance of identifying brain homogenate within the cell at any one time point is reduced, and the numbers reported here would be artificially low. However, this scenario would not be expected, based on a report that the uptake and transfer of 600- to 750-nm latex particles across M cells occurred over 90 min after contact with the FAE in a rabbit intestinal loop model (67). Second, the transport of brain homogenate through M cells could involve the phagocytosis and movement of very small particulate matter, some of which could be beyond the resolution of a light microscope (∼0.2 μm) and therefore undetectable (65). The reported diameter of transported microparticles within M cells in other studies is 0.02 to 10 μm (22); therefore, we may have been unable to identify the smaller range of particulate brain homogenate that was contained within the M cells. Regardless of resolution issues, definite identification of transcellular transport of prions within M cells will require ultrastructural analysis. While the relative contributions of the two routes identified in this report to pathogenesis and the development of disease remain to be determined, the apparently lesser role of M cell involvement in prion transepithelial transport reported here is consistent with that in with sheep gut-loop model of prion infection and in sheep orally inoculated with prions (2, 38, 46), where the FAE and M cells do not appear to be involved in the transport of prion proteins across the gut wall.

The results of this study indicate that PrP^d can gain access to lymphatic vessels quickly and that transport across the nasal mucosa and into the vessels persists for as long as 3 h after inhalation. It is not likely that the persistence of brain homogenate in the lymphatics is simply due to a low rate of lymph flow in the nasal cavity, given that flow rates have been reported to average 3 to 9 μm/s in different species (5, 23, 82) and that dye placed in the nose reaches cervical lymph nodes in 14 to 51 min, depending on the species (88). The presence of brain homogenate within lymphatics is consistent with their role of draining fluid and macromolecules from the extracellular matrix of the lamina propria in the nasal cavity. Material located in the lamina propria is propelled toward lymphatic capillaries by interstitial fluid pressure and enters the capillaries, which are made permeable by the lack of a basement membrane and the presence of anchoring filaments (for a review, see reference 81). Thus, it appears that the lymphatics, and subsequently the circulatory system, can serve as a route for the systemic dispersion of prions that have been inhaled into the nasal cavity. Consistent with this pattern of transepithelial transport following inhalation was the notable lack of brain homogenate or PrP^d adjacent to, or within, the NALT in all the animals examined in this study. While it is possible that direct neuroinvasion of prions may occur via somatic and autonomic nerves located in the lamina propria of the nasal cavity, this has not been reported.

ACKNOWLEDGMENTS

We sincerely thank Jacob Ayers, Albert Lorenzo, Melissa Clouse, Shawn Feilmann, and Theresa Lonmeth for excellent technical assistance with this work.

This work was supported by the National Institute of Neurological Disorders and Stroke (RO1 NS061994 and RO1 NS061994-03S1), Health Future Foundation Discretionary Award 200600-713131d, and grant G20RR024001 from the National Center for Research Resources.

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

Footnotes

Published ahead of print 12 September 2012

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PERMALINK

Rapid Transepithelial Transport of Prions following Inhalation

Anthony E Kincaid

Kathryn F Hudson

Matthew W Richey

Jason C Bartz

Abstract

INTRODUCTION