Experimental scrapie in 'plt' mice: an assessment of the role of dendritic-cell migration in the pathogenesis of prion diseases

Transmissible spongiform encephalopathies (TSE) are neurodegenerative disorders that can be transmitted by unconventional infectious agents termed prions. The essential component of prions is an abnormal conformer of the host-encoded prion protein PrP. This unique infectious property is the cause of natural epidemics that affect several mammalian species including sheep, cows, deers and humans (review in Prusiner, 2001; McKintosh et al., 2003; Mabbott & MacPherson, 2006; Aguzzi & Heikenwalder, 2006). Experimental models have been developed by passing natural TSE agents into rodents, especially the mouse, thus providing tools of considerable interest for pathophysiological studies. After oral or transcutaneous peripheral inoculation, the disease features a strikingly long incubation period during which infectivity and pathological prion protein (PrP^Sc) accumulate in secondary lymphoid organs (review in Aucouturier & Carnaud, 2002). This progressive increase of infectious titres in lymphoid tissues seems critical for further neuroinvasion (Lasmezas et al., 1996; Klein et al., 1997). Immobile anatomical elements, such as germinal centre-associated follicular dendritic cell (FDC) networks (Brown et al., 1999) and peripheral nervous endings (Glatzel et al., 2001; Race et al., 2000), were shown to accumulate prions at the periphery early in the pathogenesis. However, the mechanism as to how prions are transported from the mucosa or skin to lymphoid follicles and to sites of neuroinvasion remains to be explained. In CCR5-knockout mice, the shortened distance between FDC and nerve terminals results in accelerated neuroinvasion (Prinz et al., 2003). Yet the mechanism(s) of prion trafficking in normal lymphoid tissues is unclear, as well as the way they rapidly propagate to distant lymphoid organs.

Among circulating immune cells, blood lymphocytes were found to bear little or undetectable amounts of prion infectivity (Raeber et al., 1999). Recent studies have pointed out a possible role of haematopoietic dendritic cells (DCs) in the uptake and transport of prions. Indeed, DCs display unique functions of antigen capture and migration from sites of entry to secondary lymphoid organs (review in Randolph et al., 2005), making them appropriate candidates for prion dissemination. Certain observations in natural and experimental TSE support the possibility of DC involvement: in sheep scrapie, PrP^Sc was detected in cells of the Peyer's patches expressing CD68, a marker of the mononuclear phagocyte lineages (Andreoletti et al., 2000); another study revealed infected DCs in the mesenteric lymph shortly after oral administration of scrapie-associated fibrils to rats (Huang et al., 2002); PrP^Sc-positive DCs and macrophages were demonstrated in vessel walls of CreutzfeldtJakob disease patients (Koperek et al., 2002). Finally, intravenously injected DCs proved sufficient to provoke scrapie in immunodeficient mice (Aucouturier et al., 2001) and were even detected in the brain parenchyma of certain prion-infected mice (Rosicarelli et al., 2005). However, although DCs are involved in early stages of prion infections, their actual contribution to TSE pathogenesis remains to be evaluated.

Immature DCs are continually recruited in the mucosa and skin for sampling antigens. Following interaction with antigens and activation, they express membrane chemokine receptor CCR7 and become able to migrate to secondary lymphoid organs. The paucity of lymph node T cells (plt) mutation occurs on mouse chromosome 4 and results in recessive loss of expression of both functional CCL19 and CCL21-Ser genes (Nakano et al., 1998). CCL19 and CCL21 are natural ligands of CCR7 involved in the migration of naive T cells and activated DCs into T-cell zones of lymphoid organs. As a consequence of deficient expression of these chemokines, the number of DCs in the lymph nodes of plt mice is reduced approximately threefold and their migration after contact sensitization is strongly affected (Gunn et al., 1999; Vassileva et al., 1999). While the total number of DCs in the spleen remains normal in plt C57BL/6 mice, their distribution is altered due to deficient migration from the periphery and into the white pulp. Except for deficient T-cell zones, plt mice display normal lymphoid histological organization in spleen, lymph nodes and gut-associated lymphoid tissue; they have no impairment of germinal centre reaction and the lymphoid microarchitecture is not disturbed (Mori et al., 2001 and unpublished personal data). Thus, the plt mouse represents an interesting model for studying DC migration from peripheral mucosa and skin to lymphoid follicles (Randolph et al., 2005).

In an attempt to evaluate the participation of DCs in prion spreading, we infected plt mice with two scrapie strains using different routes of inoculation. While viable DCs accelerate disease onset as compared with killed DCs, the plt mutation has little or no effect on scrapie pathogenesis.

Animals.
Mice carrying the plt mutation were back-crossed 10 times onto the C57BL/6 background. plt and wild-type C57BL/6 mice were housed in individual ventilated cages under strict specific-pathogen-free conditions, in compliance with European recommendations. In all experiments, female mice were 6 to 8 weeks old at inoculation.

Disease monitoring.
Mice were monitored twice a week for clinical disease starting at 10 weeks after infection, by observing their activity levels and competence on a set of parallel bars as described previously (Aucouturier et al., 2001).

Determination of the infectious titres in brain homogenates and cell suspensions.
Serial log₁₀ dilutions of brain pool homogenates from mice infected with either the 139A or the ME7 scrapie strain were inoculated intracerebrally into 8-week-old C57BL/6 mice. Doses leading to 50 % infection by intracerebral inoculation (ID₅₀) were calculated for each inoculum using the SpearmanKärber method.

DCs were inoculated similarly by the intracerebral route and survival periods were used to deduce the titre of cell suspensions from brain titration curves.

Immunofluorescence studies of lymphoid structures.
DC distribution in spleen was revealed on frozen sections with phycoerythrin-conjugated anti-CD11c monoclonal antibody (clone N418; Pharmingen). Preparations were counterstained with DAPI (Sigma-Aldrich).

For the analyses of nerve fibres and endings, cryosections of Peyer's patches, mesenteric lymph nodes and spleens were incubated for 1 h at room temperature with rabbit primary antibodies directed to intermediate neurofilaments subunits M (150 kDa) and L (68 kDa) (Chemicon), glial fibrillary acid protein (GFAP) (Dako) and synaptophysin (Chemicon), and revealed with a tetramethyl-rhodamine-conjugated goat anti-rabbit secondary antibody (Molecular Probe). Negative controls without primary antibodies were tested. Immature B-cells were immuno-stained by fluorescein isothiocyanate-labelled rat anti-IgD antibody (BD-Biosciences). All samples were observed with a Leica TCS SP2 confocal microscope. Ten micrometre cryosections were scanned on their best fluorescent zone (57 µm), which was divided into 15 sections.

Inoculation of C57BL/6 mice with scrapie-infected DCs.
CD11c+ DCs were isolated from spleens of 139A-infected mice at 1012 weeks post-inoculation (p.i.) by positive magnetic cell sorting (Miltenyi Biotec) as described previously. Infectivity titres borne by purified DCs were similar to our previous results (Aucouturier et al., 2001) (Table 1). One group of C57BL/6 mice was inoculated with 10⁶ live infected DCs (corresponding to 10^4.63 ID₅₀ by intracerebral route) in the rear footpads, and another group was inoculated similarly with 10⁶ DCs from the same preparation that had been killed previously by three cycles of freezingthawing. At weeks 1 and 3 p.i., one mouse of each group was sacrificed and PrP^Sc accumulation was investigated by immunohistochemistry on lymphoid tissues (popliteal, inguinal and mesenteric lymph nodes and spleen).

Table 1. Infectivity titres of scrapie-infected tissues and DCs, as measured by intracerebral inoculations of C57BL/6 mice

Inoculation of plt mice.
Homozygous, heterozygous plt and wild-type mice were inoculated intraperitoneally with 100 µl of a 0.1 % (corresponding to 10^3.63 ID₅₀ by intracerebral route) titrated 139A-scrapie brain homogenate. Two animals per group were sacrificed at week 25 p.i., and spleens were analysed by immunofluorescence and immunohistochemistry. The remaining mice were followed-up for clinical scrapie by observing their activity levels and competence on a set of parallel bars, as described previously (Aucouturier et al., 2001).

Homozygous and heterozygous plt mice were inoculated subcutaneously in the rear footpads with 2x40 µl of 0.2 % (10^3.83 and 10^4.11 ID₅₀ for 139A and ME7, respectively) or 0.01 % (10^2.53 and 10^2.81 ID₅₀) 139A- or ME7-titrated brain homogenates (Table 1). In order to evaluate the early stages of scrapie propagation, two mice of each group were sacrificed at weeks 2 and 6 p.i., and lymphoid tissues (popliteal, inguinal and mesenteric lymph nodes and spleen) were examined for PrP^Sc accumulation. The remaining mice were kept for clinical studies.

For oral inoculations, heterozygous and homozygous plt mice were starved for 24 h and then fed with 200 µl of a 139A- or ME7-infected brain homogenate at 10 % (10^5.93 and 10^6.2 ID₅₀, respectively) in Intralipid as described by Maignien et al. (1999). One mouse of each group was sacrificed at days 60 and 120 p.i., and spleen, Peyer's patches, mesenteric lymph nodes and brain were collected. Clinical follow-up was performed with the remaining mice.

Finally, homozygous and heterozygous plt mice were inoculated intracerebrally with 20 µl of 0.01 % (10^1.93 ID₅₀) or 1 % (10^3.93 ID₅₀) titrated 139A-scrapie brain homogenate.

PrP^Sc immunohistochemistry.
Immunodetection of PrP^Sc was performed on 5 µm tissue sections from 4 % paraformaldehyde fixed organs. Rehydrated sections were treated for 5 min with 98 % formic acid, autoclaved for 10 min at 121 °C in 10 mM citrate buffer, pH 6.1, and digested with 10 µg proteinase K (Sigma-Aldrich) ml¹ for 15 min at 37 °C. Endogenous peroxidase was blocked with 0.3 % H₂O₂. Sections were saturated with 20 % goat serum, then incubated for 60 min with anti-PrP monoclonal antibody (clone SAF83, kindly provided by Jacques Grassi, CEA Saclay, France) and 30 min with the peroxidase-conjugated EnVision reagent (K4000; Dako). Each step was followed by washes with 1 % skimmed milk and 0.05 % Tween 20 in PBS. Sections were counterstained with Mayer's haematoxylin. Negative controls were performed using tissue sections from non-infected mice and PrP^Sc-positive tissue sections without primary antibody.

Influence of DC vitality on scrapie progression from the skin
Bioassay measurement confirmed that splenic DCs isolated from infected mice bore high infectivity titres (Table 1). To evaluate their ability to propagate scrapie agent from a distal subcutaneous site, we inoculated these cells in the rear footpads, either live or killed by freezingthawing cycles. All tested lymphoid organs were negative for PrP^Sc staining at week 1 p.i., and a few lymphoid follicles stained for PrP^Sc in draining popliteal lymph nodes at week 3 p.i. in both groups (data not shown). Although PrP^Sc immunohistochemistry may hardly be quantitative, there was no evident difference between mice that had received a live or killed inoculum. However, the incubation periods of mice inoculated with live DCs were significantly shorter (mean+SD=203+10 days, n=20) than those observed after injection of killed DCs (221+29 days, n=19, P=0.003, MannWhitney test) (Fig. 1). This difference remained highly significant (P=0.008) even after excluding two mice of the latter group that displayed particularly long (287 and 311 days) incubation periods.

(12K): Fig. 1. Comparison of incubation periods of C57BL/6 mice after distal subcutaneous inoculation with live (CD11c+) or killed (kCD11c+) infected DCs (MannWhitney test).

Comparison of innervation of lymphoid tissues in plt and normal C57BL/6 mice
The topography of the peripheral nervous system was compared in Peyer's patches, mesenteric lymph nodes and spleen from plt and wild-type mice. plt and wild-type mice displayed identical patterns of innervation in all tested areas (data not shown): within the gut-associated lymphoid tissue, numerous nerve fibres were detected in the villi and crypts with antibodies directed against neurofilaments (NF) 68 kDa subunit, GFAP and synaptophysin. Few nerve fibres stained for GFAP in the subepithelial dome, and for NF 150 and 68 kDa subunits in the interfollicular regions. In mesenteric lymph nodes, nerve fibres of the interfollicular region of the cortex were slightly positive for NF 68 kDa subunit, GFAP and synaptophysin. Comparable results were observed in the T-cell zone except for the absence of detectable synaptophysin-positive fibres, while nerve endings stained for NF 150 kDa subunit. In spleens, T-cell zones that surrounded the central arteriole displayed nerve fibres positive for all tested markers. White pulp-associated vessels were labelled by anti-NF 68 kDa. The marginal zones surrounding the lymphoid follicles and T-cell zones were positive for NF 68, 150 kDa and GFAP. In all studied lymphoid tissues, no nerve fibres were detected in germinal centres of lymphoid follicles, with the nearest nervous elements being at the same distances from FDC networks in both plt and control mice.

Scrapie pathogenesis in plt mice after intraperitoneal and subcutaneous inoculation
plt mutants have a natural defect in CCL19- and CCL21-chemokine expression, leading to an altered migration of DCs to T-cell zone areas of lymphoid follicles (Gunn et al., 1999). Indeed, lower amounts of CD11c+ DCs were found in homozygous plt mice as compared with heterozygous and wild-type littermates (Fig. 2ac). Although similar amounts of PrP^Sc were detected in spleen lymphoid follicles from wild-type mice, homozygous and heterozygous plt mice 25 weeks after intraperitoneal inoculations with 100 µl 0.1 % 139A scrapie brain (Fig. 2df), the incubation time differed significantly between groups (means 180, 179 and 190 days for wild-type, plt/+ and plt/plt, respectively, P=0.003, non parametric KruskalWallis test, see Table 2). Comparisons of pairs revealed that the clinical onset was similar in heterozygous plt and wild-type mice, and significantly delayed by 11 days in homozygous plt mice. Since wild-type and heterozygous plt mice displayed similar numbers of DCs in lymphoid follicles, and identical scrapie lymphoinvasion and clinical profiles, heterozygous plt mice were used as controls in further experiments.

(92K): Fig. 2. Immunohistochemical analysis of spleens from wild-type (a, d), heterozygous (b, e) and homozygous (c, f) plt mice 25 weeks after intraperitoneal infection with 100 µl 0.1 % 139A brain homogenate. Absence of red staining for CD11c in the T-cell zone of homozygous plt mice (c), as compared to normal (a) and heterozygous plt (b) mice, where DCs were normally found in the T-cell area. PrPSc accumulation (brown staining) in lymphoid follicles was similar in wild-type, plt/+ and plt/plt mice (d, e and f, respectively) (magnification: x400).

Table 2. Kinetics of scrapie pathogenesis in plt and control mice, after intraperitoneal (ip), subcutaneous (sc), oral and intracerebral (ic) inoculations

We next compared the progression of scrapie in homozygous and heterozygous plt mice after subcutaneous inoculation of low doses of 139A or ME7 scrapie agent in the footpads; a 20-fold higher dose of 139A agent was also tested. In order to better analyse the infection of lymphoid tissues, animals were sacrificed at early times, i.e. 2 and 6 weeks p.i. As shown in Fig. 3(af), no evident difference of PrP^Sc accumulation could be detected between plt and control heterozygous mice, whatever the dose and strain. Statistical analyses revealed slightly longer durations of clinical disease in plt mice that received a low dose of ME7 (P=0.05, MannWhitney test) or a high dose of 139A scrapie (P=0.04). On the other hand, no significant difference of incubation periods and survivals was found between homozygous and heterozygous mutants (Table 2).

(52K): Fig. 3. Similar PrPSc (brown staining) accumulation levels and PrPSc-positive follicle numbers were found in spleens from heterozygous (ac) or homozygous (df) plt mice 6 weeks after subcutaneous inoculation of ME7 (a, d) or 139A scrapie strains (b, c, e, f) using a low (0.01 %, a, b, d, e) or high dose (0.2 %, c, f). PrPSc accumulation in Peyer's patches (g, i) and spleen (h, j) was also similar at day 60 after oral inoculations of heterozygous (g, h) and homozygous (i, j) plt mice with 10 % ME7 brain (original magnification x100).

Scrapie pathogenesis in plt mice after oral inoculation
Because the oral route is a frequent way of natural TSE infections with specific pathways of prion propagation, we also studied experimental scrapie in plt mice after oral inoculation. An important individual variability of lymphoinvasion and clinical course was observed when using relatively low doses (100 µl 2 % scrapie brain, corresponding to 10^4.93 ID₅₀ by intracerebral route), especially with 139A (data not shown), but the use of 20-fold higher doses yielded reproducible results. Under such conditions, similar PrP^Sc accumulations were demonstrated at 60 days p.i. in Peyer's patches, mesenteric lymph nodes and spleens from heterozygous and homozygous plt mice (Fig. 3gj), and no significant difference in clinical course was observed, whatever the scrapie strain used (Table 2).

Intracerebral scrapie inoculation
Because clinical stage duration was slightly increased in plt mice using certain conditions of scrapie inoculation by the peripheral route, and because chemokines such as CCL21 are known to participate in neuroinflammatory conditions (Columba-Cabezas et al., 2003; Dijkstra et al., 2004), we looked for a possible involvement of CCL19 and/or CCL21 in neuropathogenesis by inoculating scrapie directly into the brain. No significant difference was found in the durations of incubation and clinical stages between wild-type, heterozygous and homozygous plt mice inoculated with a low dose of 139A (KruskalWallis non-parametric test). A 100-fold higher dose (1 % scrapie brain homogenate) yielded similar clinical courses in heterozygous and homozygous plt mice (Table 2).

DCs have unique functional properties of capturing pathogens in the mucosa and skin, and then become able to migrate to secondary lymphoid organs where they present processed antigens to T cells. Here, we confirmed that spleen DCs isolated from scrapie mice 1012 weeks p.i. display remarkably high infectivity titres (Aucouturier et al., 2001). Thus, in spite of their ability to degrade prions in certain in vitro models (Luhr et al., 2002; Rybner-Barnier et al., 2006), spleen DCs do contain significant amounts of infectious material. We next attempted to assess the importance of their migration function in prion spreading from peripheral sites of inoculation.

In mice inoculated subcutaneously at a distal site with infected DCs, the incubation was shortened by 18 days when cells were alive, as compared with killed cells. This significant difference suggests that DCs may be actively involved in scrapie development. Whether this effect relates to DC migration, or to a proper ability of these cells to amplify TSE agents, or to any other mechanism, remains to be elucidated. A local inflammatory reaction due to injection of cells could introduce a bias in our results (Heikenwalder et al., 2005); however, since it is well recognized that necrotic debris (rather than live syngenic cells) are prone to activate pattern recognition receptors (Matzinger, 2002; Medzhitov & Janeway, 2002) this would have logically resulted in an effect opposite to what we observed. Thus, as described with viruses such as human immunodeficiency virus (see review in Teleshova et al., 2003), even though DCs entrap TSE agents, they not only seem insufficient to control infection but could also be a vector of propagation.

PrP^Sc accumulations in draining lymph nodes looked similar at 3 weeks p.i. in mice inoculated with live and killed scrapie cells. More quantitative analyses of lymphoid tissue infection would be necessary for evaluating the impact of DCs on pre-clinical stages of scrapie, but it seems that prion lymphoinvasion is not strongly dependent on DC functions. In line with this hypothesis, immunohistochemical analyses revealed normal kinetics of lymphoinvasion in plt mice, whatever the dose and scrapie strain. According to the present results, CCL19/CCL21-dependent DC migration to T-cell zones does not appear as a determining factor of prion accumulation in lymphoid organs. In mice with a deficient migration of Langerhans cells, an epidermal subset of DCs, levels of PrP^Sc deposits in draining lymph nodes were also unaffected as compared with controls (Mohan et al., 2005). Our own results from transcutaneous and oral scrapie inoculations indicate that CCL19/CCL21-dependent migration of all mucosa and skin DC subsets does not significantly participate in the lymphoinvasion process.

Clinical follow-up of orally inoculated mice revealed no difference between plt and controls. Nonetheless, we observed some differences in the clinical time-course between plt and control mice under certain conditions of peripheral transcutaneous inoculation. In plt mice, the incubation was ∼10 days longer after intraperitoneal inoculation with a high dose of 139A, and the clinical stage was slightly prolonged with a low dose of 139A or a high dose of ME7 by subcutaneous route. Such differences were not found in other tested conditions. Because these observations apparently related to late rather than early pathogenetic events, they could be caused by chemokine defects in the brain of plt mice. Indeed, several studies pointed to the involvement of CCL21 in neuroinflammatory disorders such as multiple sclerosis and experimental autoimmune encephalomyelitis (Columba-Cabezas et al., 2003; Pashenkov et al., 2003). CCL21 is the only known chemokine that also binds a receptor from another chemokine receptor family, CXCR3. The latter is expressed by microglial cells and allows them to be recruited and activated in response to neuronal death (Rappert et al., 2002). In this study, direct injection of prions into the brain did not reveal any difference in clinical course between normal and plt mice, apparently ruling out a role of brain CCL21 (or CCL19) in scrapie neuropathology. Studies of scrapie susceptibility in CCR7- and CXCR3-knockout mice could help delineate more precisely the possible involvement of these chemokines.

It is difficult to exclude that some defect of the plt mutation, unrelated to DC migration, could interfere with scrapie pathogenesis. Immunohistological analyses revealed no difference between innervation patterns of lymphoid tissues from plt and normal mice. It remains possible that the T-cell defect in interfollicular regions would play a role; however, these cells are unlikely to be involved in scrapie (Klein et al., 1997; Raeber et al., 1999; Aucouturier & Carnaud, 2002).

Finally, Henning et al. (2001) showed that the migration defect in plt mice could be restored by the immunosuppressive drug FTY720, suggesting that other mechanisms could compensate for the CCR7-ligand deficiency. Thus, whether prion-infected DCs might undertake unusual migration pathways is a possibility that remains to be explored.

In conclusion, the CCL19/CCL21-mediated DC migration seems not to significantly influence the early scrapie pathogenesis. Still, some facilitating effect is provided by DCs, as shown by inoculation of live infected cells. This effect seems not to be related to prion lymphoinvasion, suggesting that DCs are able to propagate infection directly to sites of neuroinvasion or use other means of propagation (Rosicarelli et al., 2005). In our opinion, further studies on DCs in TSE pathogenesis should be aimed at understanding their interactions with the nervous system and evaluating the potential benefits of blocking this interaction.

This work was supported by European Community contract QLK5-CT-2002-01044. We thank all partners of the ImmunoTSE network for helpful discussions, Isabelle Renault and Fabienne Cortade for mouse breeding, Sandrine Melo for technical assistance and Martine Bruley Rosset for critical reading of the manuscript.

Footnotes

^,†, Gauthier Dorban³, Hideki Nakano⁴, Terutaka Kakiuchi⁵, Claude Carnaud¹, Pierre Sarradin⁶ †Present address: Centre for Research in Vascular Biology, BioSciences Institute, University College, Cork, Ireland. ‡Present address: INRA, UE1277, Plate-Forme d'Infectiologie Expérimentale, PFIE, 37380 Nouzilly, France.