Scrapie Agent Neuroinvasion in Aged Mice Dendritic Cell Status ...

The Journal of Immunology

The Effects of Host Age on Follicular Dendritic Cell Status Dramatically Impair Scrapie Agent Neuroinvasion in Aged Mice1 Karen L. Brown,2* Gwennaelle J. Wathne,* Jill Sales,† Moira E. Bruce,* and Neil A. Mabbott2* Following peripheral exposure, many transmissible spongiform encephalopathy (TSE) agents accumulate first in lymphoid tissues before spreading to the CNS (termed neuroinvasion) where they cause neurodegeneration. Early TSE agent accumulation upon follicular dendritic cells (FDCs) in lymphoid follicles appears critical for efficient neuroinvasion. Most clinical cases of variant Creutzfeldt-Jakob disease have occurred in young adults, although the reasons behind this apparent age-related susceptibility are uncertain. Host age has a significant influence on immune function. As FDC status and immune complex trapping is reduced in aged mice (600 days old), we hypothesized that this aging-related decline in FDC function might impair TSE pathogenesis. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during their lifespans, although most mice displayed histopathological signs of TSE disease in their brains. Our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Furthermore, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population. The Journal of Immunology, 2009, 183: 5199 –5207.

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ransmissible spongiform encephalopathies (TSEs),3 or “prion diseases”, are subacute neurodegenerative diseases that affect both humans and animals. In the TSE-affected host, pathology appears to be restricted to the CNS and characteristically includes neuronal loss, spongiform pathology, glial activation, and amyloidal aggregations of an abnormally folded host protein. The host prion protein (PrPc) is widely expressed in both humans and animals, and its expression is crucial for TSE disease susceptibility. During TSE disease, changes occur to the secondary and tertiary conformation of PrP, dramatically affecting the physicochemical and biological properties. This disease-specific isoform of the prion protein, termed PrPSc, accumulates in TSE-affected tissues in abnormal, detergent-insoluble, relatively proteinase-resistant aggregates. The nature of the TSE agent is uncertain, but infectivity co-purifies with PrPSc in diseased tissues and is a useful biochemical marker for the TSE agent (1). The “prion hypothesis” argues that PrPSc constitutes a major, or the sole, component of infectious agent and facilitates conversion of PrPc to PrPSc. *The Roslin Institute and Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Roslin, United Kingdom; and †Biomathematics & Statistics Scotland, Edinburgh, United Kingdom Received for publication August 19, 2008. Accepted for publication August 13, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by funding from the European Commission, UK Biotechnology and Biological Sciences Research Council, and the Medical Research Council.

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Address correspondence and reprint requests to Dr. Karen L. Brown and Dr. Neil A. Mabbott, The Roslin Institute and Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Roslin, Midlothian EH25 9PS, U.K. E-mail: karen.brown@ roslin.ed.ac.uk and [email protected]

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Abbreviations used in this paper: TSE, transmissible spongiform encephalopathy; FDC, follicular dendritic cell; PrP, prion protein; vCJD, variant Creutzfeldt-Jakob disease; BSE, bovine spongiform encephalopathy; CR, complement receptor; i.c., intracerebral; TH, tyrosine hydroxylase; PK, proteinase K. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802695

Some TSE agents, including natural sheep scrapie, bovine spongiform encephalopathy (BSE), chronic wasting disease in mule deer and elk, and kuru and variant Creutzfeldt-Jakob (vCJD) disease in humans, are acquired by peripheral exposure (e.g., orally or via lesions to skin or mucous membranes). Experimental studies in mice (2– 4) and analysis of tissues from sheep with natural scrapie (5), mule deer fawns with chronic wasting disease (6), and a patient with vCJD (7) suggest that after oral exposure TSE agents accumulate first upon follicular dendritic cells (FDCs) in B cell follicles within the GALT as they make their journey from the site of infection to the CNS (a process termed neuroinvasion). FDCs are a distinct lineage from conventional hemopoietic dendritic cells, as they are considered to derive from stromal precursor cells, are nonphagocytic, and are nonmigratory (8). In mouse TSE models, agent accumulation upon PrPc-expressing FDCs within the lymphoid tissues is critical for disease pathogenesis (9, 10), as in their absence, neuroinvasion is significantly impaired (2, 11, 12). From the lymphoid tissues, translocation to the CNS occurs via the peripheral nervous system (13, 14). Strong evidence indicates that the BSE epidemic in the United Kingdom most probably occurred and was sustained via the feeding of BSE-contaminated ruminant meat and bonemeal to cattle (15). Furthermore, the consumption of BSE-contaminated meat products is considered the most likely source of the human TSE disease vCJD (16). During the United Kingdom BSE epidemic it was estimated that almost 500,000 infected cattle were slaughtered for human consumption (17). Despite this probable widespread exposure of the United Kingdom human population to the BSE agent, clinical cases of vCJD have predominantly occurred in young people (17). The possibility that young adults were exposed to greater levels of BSE by dietary preference has not been substantiated (18), suggesting that other age-related factors may influence susceptibility to peripherally acquired TSE agents. FDCs characteristically trap and retain Ags, storing them on their surfaces for long periods (8). Ags are retained on FDCs in the

5200 form of immune complexes comprising Ag, Abs, and/or complement components. These complexes bind to FDCs via complement receptors (CRs) in naive mice and also Ab Fc receptors in immunized mice (19). Complement components C1q and C3, as well as cellular CRs, are likewise considered to play an important role in the localization of TSE agents to FDCs (20 –22). In aged (600 days old) mice, FDC status is significantly impaired. Aged FDCs appear atrophic and have a reduced capacity to trap and retain immune complexes, leading to deficits in Ab responsiveness and germinal center formation (23–26). Therefore, using an experimental mouse TSE pathogenesis model (mouse-passaged scrapie agent) experiments were designed to determine whether the decline in FDC status in aged mice would influence the early accumulation of the TSE agent within lymphoid tissues and subsequent neuroinvasion.

Materials and Methods Mice C57BL/Dk mice were aged to ⬃600 days under normal specific pathogenfree conditions before exposure to the scrapie agent. Six- to 8-wk-old C57BL/Dk mice were used as young adults. All experimental procedures were approved by the Institute’s ethical review committee and were conducted according to the strict regulations of the U.K. Home Office Animals (Scientific Procedures) Act 1986.

Scrapie agent exposure and disease monitoring Where indicated, mice were injected i.p. or intracerebrally (i.c.) with 20 ␮l of a 1% (w/v) scrapie brain homogenate prepared from mice terminally affected with the ME7 scrapie agent strain (containing ⬃1 ⫻ 104 i.c. ID50 U). For oral exposure, mice were fed individual food pellets doused with 50 ␮l of a 10% (w/v) dilution of terminally affected scrapie brain homogenate. Following scrapie agent exposure, mice were coded and assessed weekly for signs of clinical disease and culled at a standard clinical endpoint (27). Survival times were recorded for mice that did not develop clinical signs of disease and were culled when they showed signs of intercurrent disease. Scrapie diagnosis was confirmed by histopathological assessment of vacuolation in the brain. For the construction of lesion profiles, vacuolar changes were scored in nine gray matter areas of brain as described (28). At the times indicated, some mice were culled and tissues taken for further analysis. For bioassay of scrapie agent infectivity, individual spleens were prepared as 10% (w/v) homogenates in physiological saline. Groups of 10 –12 C57BL/Dk indicator mice were injected i.c. with 20 ␮l of each homogenate. A dose/incubation period response curve was prepared for ME7 scrapie agent-infected spleen tissue titrated serially in C57BL/Dk mice (supplemental Table I and supplemental Fig. 1).4 The scrapie titer in each young and aged spleen assayed was then estimated from the mean incubation period in each group of indicator mice, by reference to the dose/incubation period response curve using the relationship y ⫽ 13.879 ⫺ 0.0506x (where y indicates the log ID50/20 ␮l of homogenate; x, incubation period time; supplemental Table II).

Immunohistochemistry For the detection of disease-specific PrP (PrPd) in brain and spleen, tissues were fixed in periodate-lysine-paraformaldehyde fixative and embedded in paraffin wax. Sections (thickness, 6 ␮m) were deparaffinized and pretreated to enhance the detection of PrPd by hydrated autoclaving (15 min, 121°C, hydration) and subsequent immersion in formic acid (98%) for 5 min (29). Sections were then immunostained with 1B3 PrP-specific polyclonal antiserum (30). Tyrosine hydroxylase (TH)-expressing sympathetic nerves were detected using TH-specific polyclonal rabbit antiserum (Chemicon Europe). Immunolabeling was revealed using alkaline phosphatase conjugated to the avidin-biotin complex (Vector Laboratories). PrPSc was detected on paraffin-embedded sections of brain and spleen by paraffinembedded tissue immunoblot analysis as described (31). Following processing, membranes were immunostained with 1B3 PrP-specific polyclonal antiserum. For the detection of FDCs, spleens were snap-frozen in liquid nitrogen. Frozen sections (thickness, 10 ␮m) were fixed in acetone, and FDCs were visualized by staining with mAb FDC-M2 to detect complement component C4 and mAb 7G6 to detect CR2/CR1 (CD21/CD35; BD Pharmingen). Cellular PrPc was detected using 1B3 PrP-specific polyclonal antiserum. 4

The online version of this article contains supplemental material.

EFFECT OF HOST AGE ON SCRAPIE PATHOGENESIS Following the addition of primary Abs, species-specific secondary Abs coupled to Alexa Fluor 488 (green) or Alexa Fluor 594 (red) dyes (Invitrogen) were used.

Quantification of FDC networks For enumeration of PrPc-expressing FDC networks, coded spleen sections from aged (n ⫽ 19) and young (n ⫽ 9) mice were immunolabeled for cellular PrPc as described above and examined using a Zeiss confocal micoscope. The total number of PrPc-expressing follicles within each entire spleen section was then counted. Data are presented as the mean number of PrPc-expressing follicles per spleen section (⫾SEM). Statistical analysis was performed using Student’s t test. For analysis of C4 and PrPc colocalization in the spleens of aged and young aged mice, coded sections were double-immunostained to detect C4 and PrPc as described above. For each section, five to eight randomly selected fields of view from each spleen when viewed with the ⫻10 objective were captured. Within each captured field, the number of intact or disrupted FDC networks and the number of each type of FDC network with colocalized PrPc expression were recorded. Data are presented as the mean percentage positive FDC networks per spleen (⫾SEM). Statistical analysis was performed using Student’s t test.

Quantification of TH-positive nerve fibers in the spleen Coded spleen sections from young and aged mice were immunostained to detect TH-positive nerves as described above, and the area occupied by these cells was quantified. To do so, six fields of view from each spleen when viewed with the ⫻10 objective were captured, and the area covered by TH-positive cells in each field of view was measured using ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). Data are expressed as the mean percentage of the area occupied by TH-positive cells (⫾SEM). Statistical analysis was performed using Student’s t test.

Statistical analyses Differences in the proportions of mice having positive vacuolar pathology (young vs aged) were investigated using a Fisher’s exact two-tailed test. To compare the severity of the vacuolation between aged and young mice, the extent of the vacuolation was assessed in each of nine brain regions, with scoring done on a scale of 0 –5. As a summary measure, the areas under the lesion profile curves (AUC) were estimated using the trapezoidal rule, for those mice that could be scored in at least eight of the regions (i.p. route, n ⫽ 12 young, n ⫽ 3 aged; oral route, n ⫽ 12 young, n ⫽ 5 aged; i.c. route, n ⫽ 6 young, n ⫽ 3 aged). For each route separately, the AUC was found for each mouse using the trapezoidal rule. Differences in median AUC between the aged and young animals were tested using a two-tailed MannWhitney U test. Analysis of the incubation period data obtained from the spleen bioassay studies was conducted on all mice that showed positive pathology (n ⫽ 130). A linear mixed model was fitted to the data with status (whether the spleen came from a young or aged mouse), spleen age (how long after infection the spleen was collected) and sex of recipient mouse included as fixed factors, and spleen (n ⫽ 2 for each status per spleen age combination) fitted as a random factor. Parameters of the model were estimated using the residual maximum likelihood (REML) directive in GenStat 10th edition, and the fixed effects were tested using F tests with appropriately modified degrees of freedom.

Results Effect of host age on FDC status First we compared the status of FDCs within the spleens of young adult (6 – 8 wk old) and aged (⬃600 days old) C57BL/Dk mice by immunohistochemistry. High levels of complement component C4 in association with FDCs expressing CR2 and CR1 (CD21/35) and the cellular form of the prion protein, PrPc, were detected in the spleens of young mice (Fig. 1, A and B). The association of C4 on FDCs is considered to provide a reliable reflection of the level of immune complex trapping on these cells (24, 25, 32). The detection of complement component C4 upon most FDCs from aged mice was substantially reduced (Fig. 1, A and C), indicating that the retention of immune complexes on the FDCs was impaired. Furthermore, our analysis indicated that these networks were disrupted (Fig. 1C) and lacked the typical follicular structure observed in young mice (Fig. 1B). Expression of cellular PrPc by FDCs is critical for the accumulation of the TSE agent within the


FIGURE 1. Effect of host age on FDC status in the spleen. A, CR2/CR1 expression and bound complement component C4 and on FDCs was readily detected in the spleens of young mice (upper row, red). Although CR1/CR2 expression appeared similar in aged and young spleens (bottom row, left, red), the association of C4 upon FDCs was dramatically reduced in spleens of aged mice (bottom row, middle and right, red). Left and middle columns, original magnification ⫻40, scale bar ⫽ 10 ␮m; righthand column, original magnification ⫻200, scale bar ⫽ 50 ␮m. B, High levels of cellular PrPc expression (green) and complement component C4 (red) colocalized upon FDCs in spleens of young mice. C, Immunolabeling for PrPc and complement C4 was greatly reduced and appeared atrophied and sparsely distributed within most follicles from aged mice. D, However, some FDCs networks in spleens from aged mice displayed levels of PrPc and complement C4 similar to those observed in spleens from young mice. Original magnification ⫻400, scale bar ⫽ 100 ␮m.

spleen and efficient neuroinvasion (9, 10). In most FDC networks from aged mice, the reduced detection of complement component C4 coincided with a similar reduction in PrPc expression (Fig. 1C). However, some FDC networks from aged mice were intact and expressed levels of PrPc and FDC-associated C4 similar to those in the spleens of young mice (Fig. 1D). When we assessed the number of intact PrPc-expressing FDC networks in the spleen, highly significantly reduced numbers were observed in those from all aged mice (Fig. 2). Spleens from aged (n ⫽ 19) and young (n ⫽ 9) mice were immunolabeled with antisera specific for cellular PrPc and complement component C4. Entire sections were coded, examined microscopically, and the total number of FDC networks were counted, their status was recorded (intact or disrupted), and the numbers of each type expressing PrPc and C4 were counted. A highly significant reduction in the total number of PrPc-expressing FDC networks in the spleens of aged mice was found when compared with those from young mice ( p ⬍ 0.0003; Fig. 2A). In spleens from young mice all of the FDC networks were intact and displayed the characteristic follic-

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FIGURE 2. FDC status is significantly reduced in the spleens of aged mice. Spleens from aged (n ⫽ 19) and young (n ⫽ 9) mice were immunolabeled with antisera specific for cellular PrPc and complement component C4. Entire sections were coded, examined microscopically, and the total numbers of FDC networks were counted, their status was recorded (intact or disrupted), and the numbers of each type expressing PrPc and C4 were counted. A, A highly significant reduction in the total number of PrPc-expressing FDC networks in the spleens of aged mice was found when compared with those from young mice (p ⬍ 0.0003). B, Although a small number of intact FDC networks were observed in spleens from aged mice, a significantly greater proportion of the FDCs networks were disrupted (Disrupt.) and showed reduced levels of complement component C4 on their surfaces (p ⬍ 0.027). C, All of the FDC networks from young mice expressed cellular PrPc. In spleens from aged mice none of the disrupted FDC networks expressed PrPc. Only the small number of intact FDC networks expressed cellular PrPc. Each bar represents mean ⫾ SEM.

ular structure (Figs. 1B and 2B). Although a small number of intact FDC networks were observed in spleens from aged mice, a significantly greater proportion of the FDCs were disrupted ( p ⬍ 0.027; Fig. 2B). Further analysis showed that whereas all the intact FDC networks from young and aged mice expressed cellular PrPc, none of the disrupted FDC networks in aged mice expressed PrPc (Fig. 2C). Taken together, these data demonstrate that the number of intact PrPc-expressing FDC networks in the spleens of aged mice is substantially reduced. Furthermore, a significant majority of the FDC networks in aged spleens lack PrPc expression, are disrupted, and demonstrate an impaired ability to retain complement-opsonized immune complexes.

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EFFECT OF HOST AGE ON SCRAPIE PATHOGENESIS detected in aged spleens, and in many instances infectivity was undetectable or at trace levels ( p ⬍ 0.001; general linear mixed model; Fig. 4A). By 15 wk after exposure our data indicate that the levels of agent infectivity in the spleens of aged mice were ⬃100fold lower than those in the spleens from young adult mice (Fig. 4A). Spleens were also analyzed from two clinically scrapie-affected young mice and four aged mice that remained free of the signs of clinical disease at the end of the experiment (Fig. 4B). Our data show that TSE agent infectivity levels in spleens from aged mice were likewise reduced and never reached the levels observed in spleens from young mice. Furthermore, in the spleen of one aged animal only trace levels of agent infectivity were observed. These data show that the early accumulation of the scrapie agent upon FDCs is significantly reduced in the spleens of aged mice.

FIGURE 3. Effect of host age on TSE agent accumulation in the spleen. A, High levels of PrPd were detected in association with FDCs in spleens from young mice from 5 wk (not shown) and 10 wk after i.p. injection with the scrapie agent (upper row, red). In contrast, PrPd was not detected in the spleens of aged mice until 15 wk after i.p. exposure. B, Analysis of adjacent sections by paraffin-embedded tissue immunohistoblot confirmed the presence of proteinase K-resistant PrPSc (lower row, blue/black). Scale bar ⫽ 20 ␮m.

Effect of host age on the early accumulation of the scrapie agent upon FDCs The precise nature of the scrapie agent is uncertain, but PrPSc, a proteinase K (PK)-resistant form of the cellular prion protein, PrPc, copurifies with agent infectivity in diseased tissues (1). Within 10 wk after peripheral exposure of young adult mice to the ME7 scrapie agent, strong accumulations of PrPSc and agent infectivity accumulate upon FDCs within the spleen and are sustained until the terminal stages of disease (2, 10, 11). This early accumulation within lymphoid tissues is critical for efficient neuroinvasion (2, 10 –12). As PrPc expression (9, 10) and opsonizing complement components (20 –22) are critical for the accumulation of the scrapie agent upon FDCs, their reduced association with FDCs in the spleens of aged mice suggested to us that scrapie agent accumulation in aged spleens would likewise be reduced. In this study, PrPd is used to describe the disease-specific PrP accumulations detected by immunohistochemistry when PK treatment was not used (4). Heavy PrPd accumulations were detected in the spleens of all young adult mice at 5 wk (not shown) and 10 wk after i.p. injection with the scrapie agent (Fig. 3A) and were maintained until the terminal stages of disease. The distribution of PrPd within these tissues was consistent with accumulation upon FDCs (2, 10, 11). Paraffin-embedded tissue immunoblot analysis of tissue sections can be used to directly demonstrate the presence of TSE agent-specific PK-resistant PrPSc on histological sections (31). During processing, tissue sections are treated with PK to destroy all the cellular PrPc, leaving only the TSE agent-specific PK-resistant PrPSc (if present). Sites of PrPSc are then readily detected immunohistochemically using PrP-specific antisera. Paraffin-embedded tissue immunoblot analysis of adjacent sections confirmed that these PrPd accumulations contained PK-resistant PrPSc (Fig. 3B). In contrast, in the spleens of aged mice the accumulation of PrPSc appeared to be delayed and was not detected until 15 wk after exposure (Fig. 3B). We next compared scrapie agent infectivity levels in spleens from young adult and aged mice taken 5, 7, 10, and 15 wk after i.p. exposure. In spleens of young adult mice, high levels of scrapie agent infectivity were detected from 5 wk after exposure (Fig. 4A). However, significantly lower scrapie agent infectivity levels were

Effect of host age on scrapie susceptibility Next, we determined the effect of host age on scrapie susceptibility. As anticipated, all young adult mice succumbed to clinical disease following exposure to the scrapie agent by the i.p. or oral routes with incubation periods of 274 ⫾ 5 and 301 ⫾ 4 days, respectively (Table I). In contrast, none of the aged mice developed clinical signs of TSE disease during its lifespan (up to 366 days after exposure; Table I). To allow direct comparison, only aged mice that were culled after the first clinically positive case in the corresponding young adult mouse group were included in our analysis. Whereas characteristic TSE-specific vacuolar spongiform pathology was detected in the brains of all clinically scrapie-affected young mice, positive vacuolar pathology was only detected in 3 of 9 i.p. injected aged mice ( p ⬍ 0.001; Fisher’s exact twotailed test) and 5 of 12 orally exposed aged mice ( p ⫽ 0.002; Fisher’s exact two-tailed test; Table I). The average severity of the spongiform pathology within the brains of the aged mice with histopathological signs of TSE disease was significantly lower than that observed in the brains of clinically affected young mice ( p ⫽ 0.022, i.p. route; p ⫽ 0.001, oral route; two-tailed MannWhitney U test; Fig. 5, A and B). In contrast, when young and aged mice were injected with the scrapie agent directly into the brain by i.c. injection, each group was fully susceptible to disease and developed clinical scrapie with similar incubation periods (Table I). However, analysis of the magnitude of the spongiform pathology in the aged brains (n ⫽ 3) revealed it was significantly lower on average when compared with the young mice ( p ⫽ 0.024, two-tailed Mann-Whitney U test; Fig. 5C). Sympathetic innervation in spleens and intestines of young and aged mice Following accumulation upon FDCs, the scrapie agent subsequently spreads to the brain via the peripheral nervous system (13, 14). Immunohistochemical analysis suggested that host age did not appear to impair the status of TH-positive sympathetic nerves, as the location and density of these cells in the spleens of uninfected young and aged mice appeared similar (Fig. 6, A and B, respectively). Furthermore, quantitative assessment of the area occupied by TH-positive sympathetic nerves within the spleens of young and aged mice indicated that their density was similar in each group ( p ⫽ 0.9, n ⫽ 5, Student’s t test; Fig. 6C). Previous studies have shown that both the relative distance between FDCs and sympathetic nerves (33, 34), as well as their overall density (35), significantly influences the rate of neuroinvasion from the spleen. As each of these parameters was similar in the spleens of young and aged mice, our data imply that any potential effects of host age on


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FIGURE 4. Effect of host age on TSE agent infectivity accumulation in the spleen. Spleens were collected from (A) young and aged mice 5, 7, 10, and 15 wk after i.p. exposure and (B) from two clinically scrapie-affected young mice and four aged mice that remained free of the signs of clinical disease at the end of the experiment. Scrapie agent infectivity titers were determined by transmission into groups of indicator C57BL/Dk mice. Each point represents datum for an individual spleen. Data below the horizontal line indicate disease incidence in the recipient mice ⬍100% and considered to contain trace levels of infectivity. Statistical analysis of the incubation period data obtained from the spleen bioassay studies (A) was conducted on all mice that showed positive pathology (n ⫽ 130). A linear mixed model was fitted to the data with status (whether the spleen came from a young or aged mouse), spleen age (how long after infection the spleen was collected), and sex of recipient mouse included as fixed factors, and spleen (n ⫽ 2 for each status per spleen age combination) fitted as a random factor. Parameters of the model were estimated using the REML directive in GenStat 10th edition, and the fixed effects were tested using F tests with appropriately modified degrees of freedom. High levels of agent infectivity were detected in the spleens of young adult mice as soon as 5 wk after exposure. However, significantly lower agent infectivity levels were detected in the spleens of aged mice, with many containing trace or undetectable levels (p ⬍ 0.001; linear mixed model). a, One clinically negative animal culled at 210 days with possible vacuolar pathology in the brain.

the status of sympathetic nerves were unlikely to influence disease pathogenesis. Immunohistochemical analysis likewise suggested that host age did not appear to impair the status of PrPcexpressing (Fig. 6D) and TH-positive (Fig. 6E) sympathetic nerves in the small intestine, as their distribution and density appeared similar in tissues from young and aged mice. Taken together, these data suggest that the effects of host age on the ability of FDCs to acquire and accumulate the TSE agent were the major influence on neuroinvasion. Effect of host age on the accumulation of the scrapie agent within the brain and spleen at the terminal stages of disease During TSE disease, the deposition of PrPd in the brain typically precedes the vacuolar changes (36). We therefore determined whether PrPd was present in the brains of the peripherally exposed aged mice despite the absence of positive vacuolar pathology. Large accumulations of PrPd were detected in the brains and spleens of all clinically scrapie-affected young mice after i.p. injection (Fig. 7, A and D, respectively), oral exposure, and i.c. injection (data not shown). Within the brains of the clinically negative aged mice, PrPd was detected in most brains from the i.p.-injected mice (six of nine mice; Fig. 7, B and C) and all of the orally exposed mice (Table I).

Further analysis revealed that there was considerable variation in the detection of PrPd in the spleens of the clinically negative aged survivors (Fig. 7, E and F, and Table I). All of the peripherally exposed aged mice that displayed positive vacuolar pathology (except one orally exposed animal) had detectable PrPd in their brains and spleens (Fig. 7, B and E, respectively). In contrast, the aged mice that did not show signs of vacuolar pathology had detectable PrPd in the brain but not in the spleen (Fig. 7, C and F, respectively). The reasons for these differences are uncertain. In mice deficient in FDCs, neuroinvasion can occur in some mice by an FDC-independent process such as direct uptake by peripheral nerves (37–39), implying direct transfer of the TSE agent to the nervous system in some aged mice. Alternatively, it is plausible that the TSE agent initially accumulated upon FDCs, facilitating subsequent neuroinvasion, but that it was later cleared from the spleen as the status of the FDCs declined.

Discussion Here we show that the reduced status of FDCs in aged mice significantly impairs TSE agent neuroinvasion following peripheral exposure. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of

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EFFECT OF HOST AGE ON SCRAPIE PATHOGENESIS

Table I. Effect of host age on scrapie agent susceptibility and disease pathogenesis

Mouse Modela

Exposure Route

Individual Disease Incubation Periods or Survival Times (days)b

Aged

i.p.

247 247 254 261 275 282 303 310 323

Young

i.p.

Aged

Oral

Young

Oral

Aged Young

i.c. i.c.

244, 262, 262, 267, 267, 267, 267, 268, 268, 298, 298, 302 282 289 289 295 295 295 310 310 310 310 338 366 282, 282, 290, 295, 295, 295, 301, 301, 309, 314, 321, 321 168, 177, 194 163, 168, 176, 176, 176, 183

Mean Disease Incubation Period (days ⫾ SEM) or Survival Range

Clinical Disease

Vacuolar Pathology in Brain

PrPd in Brain

PrPd in Spleen

247–323 274 ⫾ 5

No No No No No No No No No 0/9c 12/12

No No No No No No Yes Yes Yes 3/9 ( p ⬍ 0.001)d 12/12

Yes No Yes No No Yes Yes Yes Yes 6/9 12/12

No No ND No No No Yes Yes Yes 3/8 12/12

282–366 301 ⫾ 4

No No No No No No No No No No No No 0/12 12/12

No Yes No No No No Yes No Yes No Yes Yes 5/12 ( p ⫽ 0.002)d 12/12

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 12/12 12/12

Yes No No No No Yes Yes Yes Yes Yes Yes Yes 8/12 12/12

179 ⫾ 8 174 ⫾ 3

3/3 6/6

3/3 6/6

ND ND

ND ND

Young C57BL/Dk mice were 6 – 8 wk old at the time of scrapie agent exposure. Aged C57BL/Dk mice were ⬃600 days old at the time of scrapie agent exposure. Only aged mice that were culled after the first positive TSE case in the corresponding young mouse group were included in the analysis. Incidence ⫽ no. animals affected/no. animals tested. Only aged mice that were culled after the first clinically positive TSE case in the young mice were included in the analysis. d Statistical differences between the numbers of aged mice and young mice with positive vacuolar pathology were determined using a Fisher’s exact two-tailed test. a b c

aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during its lifespan, although most mice displayed histopathological signs of TSE disease in their brains. In contrast, no significant differences in disease incubation period were observed following injection of the TSE agent directly into the brains of young and aged mice. Taken together, our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. These findings also suggest that although host age may present a significant barrier against TSE transmission, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population. Previous studies suggest that the Ag trapping capacity of FDCs becomes defective with aging, markedly impairing their ability to provide costimulatory signals to B cells and stimulate germinal center formation (23–26). In the present study the mAb FDC-M2, which detects complement C4 on FDC-associated immune complexes (32), was used to determine the ability of aged FDCs to trap and retain immune complexes. We show that the decline in the Ag trapping capacity of FDCs coincided with the loss of cellular PrPc expression. Host cells must express cellular PrPc to sustain TSE infection (40). Therefore, our data suggest that the combined defects in immune complex trapping and PrPc expression dramatically reduce the ability of aged FDCs to acquire and replicate TSE agents.

Following peripheral exposure to the scrapie agent many factors will act on the inoculum to aid its elimination from the host. For example, after oral exposure much will be eliminated by factors including digestion (by enzymes secreted into the gastrointestinal tract) and excretion. As a consequence, very little of the original inoculum will be available to be translocated to FDCs within lymphoid tissues. In young adult mice it is likely that a competitive state exists whereby cells such as macrophages aid the clearance of the TSE agent (41, 42), whereas FDCs act to expand the levels of the TSE agent above the threshold required to achieve neuroinvasion. Thus, as the status of FDCs declines in aged mice, our data suggest it is likely that a greater proportion of the residual fraction of the inoculum is gradually destroyed by the actions of phagocytic cells (41, 42) preventing or significantly impairing neuroinvasion. Although a significant majority of the FDC networks in the spleens of aged mice were disrupted, lacked PrPc-expression, and displayed a reduced capacity to retain complement-opsonized immune complexes, small numbers of intact PrPc-expressing FDC networks were observed. Therefore, it is plausible that in some of these aged mice, these small populations of functional FDCs present at the time of exposure were sufficient to allow a limited degree of neuroinvasion to occur for a transient period before their eventual decline. In the present study only those peripherally TSE agent-exposed aged mice that survived beyond the first clinically positive TSE case in the corresponding young mouse group were included in our


FIGURE 5. Effect of host age on TSE-specific neuropathology in the brain. Pathological assessment of the spongiform change (vacuolation) in the brains of young (closed circles) and aged (open circles) mice with positive vacuolar pathology. Vacuolation was scored on a scale of 0 –5 in nine gray matter areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septum; 8, retrosplenial and adjacent motor cortex; 9, cingulate and adjacent motor cortex. Each point represents mean vacuolation score ⫾ SEM for groups of 3–12 mice. To determine whether the severity of the vacuolation was significantly altered in aged mice, the area under the lesion profile curve (AUC) was found for each mouse using the trapezoidal rule. The differences in the median AUC between the aged and young mice were tested using a two-tailed Mann-Whitney U test. These analyses suggested that the severity of the vacuolar pathology was significantly lower in the aged mice following exposure to the scrapie agent by i.p. injection (A, p ⫽ 0.022, n ⫽ 12 young, n ⫽ 3 aged) and oral exposure (B, p ⫽ 0.001, n ⫽ 12 young, n ⫽ 5 aged). Although both aged and young mice injected i.c. with the scrapie agent developed clinical disease with similar incubation periods, the severity of vacuolar pathology was significantly lower in the brains of the aged mice (C, p ⫽ 0.024, n ⫽ 6 young, n ⫽ 3 aged).

analysis. The first positive TSE cases in the young mice were observed 244 days after i.p. exposure and 282 days after oral exposure. Although the corresponding ages of the aged mice were 844 and 882 days old (for i.p. and oral TSE-exposed mice, respectively) at these times, 9 aged mice survived between 247 and 323 days after i.p. exposure (between 847 and 923 days old), and 12 aged mice survived between 282 and 366 days after oral exposure (between 882 and 966 days old). None of these peripherally TSEexposed aged mice developed clinical signs of disease during its lifespan, although a proportion of the aged mice did display histopathological signs of TSE disease in their brains. This suggests that in some mice, neuroinvasion may have occurred independently of FDCs, albeit by a much less efficient route that did not allow the level of the TSE agent in the brain to reach the magnitude required to cause clinical disease during the host’s lifespan (37–39). As histopathological signs of TSE disease were detected

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FIGURE 6. Comparison of sympathetic innervation in the spleens (A–C) and intestines (D and E) of young and aged mice. A and B, Immunohistochemical detection of TH-positive sympathetic nerves (green) and FDCs (CR1/CR2-positive cells; red) in the spleens of uninfected young (A) and aged (B) C57BL/Dk mice. The location of sympathetic nerves (white arrow) within the spleens of both young and aged mice appeared similar and was predominantly located within the T cell areas, around the central arteriole. FO indicates location of FDC-containing follicles; original magnification ⫻200; scale bar ⫽ 100 ␮m. C, Quantitative analysis of the area occupied by TH-positive sympathetic nerves in the spleens of uninfected aged mice and young mice (n ⫽ 5). The area occupied by TH-positive nerves was measured in six random fields of view from each spleen when viewed with the ⫻10 objective. No significant difference between the area occupied by TH positive nerves in spleens from young and aged mice was observed (p ⫽ 0.9; Student’s t test). D and E, Immunohistochemical analysis of the status of PrPc-expressing (D) and TH-positive (E) sympathetic nerves in the small intestines of uninfected young and aged C57BL/Dk mice. No differences were observed in the distribution and density of sympathetic nerves (white arrow) within the intestines of both young and aged mice. Scale bar ⫽ 100 ␮m.

in the brains of many of the aged mice, the possibility cannot be excluded that if the mice had survived longer some may have developed clinical signs of disease after substantially extended incubation periods. However, all of the aged mice were at least 2.3 years old when culled. At present, no TSE-specific preclinical diagnostics are available, compounding the problems for disease treatment and eradication.

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FIGURE 7. Effect of host age on the accumulation of the scrapie agent within the brain and spleen at the terminal stage of disease. A, Large PrPd accumulations (red) were detected in brains of all clinically scrapie-affected young mice. B and C, In the brains of the i.p. injected clinically negative survivors, six of nine aged mice had evidence of PrPd in their brains, although vacuolar pathology was only found in three of these mice. Original magnification ⫻40. D, High levels of PrPd (red) were detected in association with FDCs in spleens from all clinically scrapie-affected young mice (red). However, there was considerable variation in the detection of PrPd in the spleens of the clinically negative survivors. E, All of the peripherally exposed aged mice that displayed positive vacuolar pathology in the brain had detectable levels of PrPd in their spleens. F, In contrast, PrPd was undetectable in the spleens of aged mice that did not show histopathological signs of vacuolar pathology in their brains. All panels, original magnification ⫻40, scale bar ⫽ 50 ␮m; Clin., presence of clinical signs of scrapie at the time of cull; Path., detection of spongiform pathology in the brain.

EFFECT OF HOST AGE ON SCRAPIE PATHOGENESIS effects of host age on TSE pathogenesis are most likely due to effects on FDC status dramatically impairing TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Data from the present study agree with data from a comparative study of Peyer’s patches from sheep, cattle, and humans that suggested an age-related association between Peyer’s patch development and susceptibility to natural TSE infection (51). In conclusion, although the reasons associated with the predominance of vCJD cases in young people may be complex, our data suggest that the pathogenesis of peripherally acquired TSE agents such as natural sheep scrapie, chronic wasting disease in cervids, and vCJD in humans are much less efficient in the aged than in young individuals. Although host age may represent an important barrier to the efficient transmission of peripherally acquired TSE agents, our data suggest there may be significant levels of subclinical disease in the elderly population. Furthermore, our data suggest that the tissue distribution of some TSE agents may differ significantly between the young and elderly. These data raise important issues for the development of reliable preclinical TSEspecific tests, the assessment of risk tissues, and the control of iatrogenic spread.

Acknowledgments We thank Maura Donaldson, Jill Oxley, Irene McConnell, Lorraine Gray, and Val Thomson (University of Edinburgh, U.K.) for excellent technical assistance; Patricia McBride, Christine Farquhar, and Robert Somerville (University of Edinburgh) for helpful discussion; Christine Farquhar for provision of 1B3 PrP-specific polyclonal antiserum; and Bob Fleming (University of Edinburgh) for assistance with image analysis.

Disclosures The authors have no financial conflicts of interest.

References The detections of some TSE agents in blood (43) or lymphoid tissue biopsy specimens such as tonsils, appendix (7, 44), and rectal-associated lymphoid follicles (45– 48) have each been proposed as useful methods to detect some preclinical TSE diseases. Our data presented herein revealed that there was considerable variation in the detection of the TSE agent in the spleens of the clinically negative aged survivors. Although most TSE-exposed aged mice had detectable levels of PrPd in their brains, PrPd was absent in the spleens of many mice. These data have important implications for the development of reliable preclinical diagnostics, as tests based on the detection of the TSE agent in blood or lymphoid tissues may be less sensitive when used on elderly patients and livestock species. Our data show that when the TSE agent was delivered directly to the brain, host age had no significant influence on the onset of clinical disease. Furthermore, our data show that host age did not influence the distribution of TH-positive sympathetic nerves in the spleen and small intestine. These data provide strong evidence that the effects of age observed on susceptibility to peripherally acquired TSE infection were not due to effects on the central and peripheral nervous systems. Our recent data suggest that conventional dendritic cells may play an important role in the initial translocation of TSE agents from the gut lumen to the GALT (49). Moretto and colleagues have recently shown that mucosal dendritic cells have a reduced capacity to prime T cells and secrete IL-15 (50). Whether aging likewise affects the distribution of DCs at the sites of TSE agent exposure and their ability to acquire them and deliver them to draining lymphoid tissues is not known. Therefore, although the effects of age on other factors cannot be entirely excluded, our data imply that following peripheral exposure, the

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