Rapid transepithelial transport of prions following inhalation

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.

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).

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).

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).

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.