Early Cytokine Elevation, PrPres Deposition, and Gliosis in Mouse
Scrapie: No Effect on Disease by Deletion of Cytokine Genes IL-12p40
Déborah Tribouillard-Tanvier,* Brent Race, James F. Striebel, James A. Carroll, Katie Phillips, and Bruce Chesebro
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
tive disorders that affect both humans and animals (24). Prion
diseases are characterized by spongiform degeneration of gray
matter, gliosis, and accumulation of a misfolded, partially pro-
tease resistant form, PrPres (also known as PrPSc), of the normal
cellular prion protein, PrPsen (also known as PrPC). Activation
and/or proliferation of astroglia and microglia is prominent dur-
duce inflammatory mediators such as cytokines and chemokines
in various neurodegenerative and infectious diseases, and all may
to PrPres accumulation and associated brain cell damage. However,
this neuroinflammation might also act either to increase or to de-
glial cells and in vivo in the brains of sick mice after scrapie infec-
tion (35). Despite extensive gliosis during prion disease, scrapie-
infected mice showed consistent significant elevation of only 6
cytokines (CCL2, CCL5, CXCL1, CXCL10, interleukin 1? [IL-
1?], and IL-12p40) (35). The low number of increased cytokines
pattern seen in the brain following a typical bacterial or viral in-
fection (3, 13, 31).
In the current experiments, to determine the cause-and-effect
process, we studied mice at a series of time points starting at 20
crogliosis, and cytokine levels in the brains of scrapie-infected mice
rion diseases, also known as transmissible spongiform en-
cephalopathy (TSE) diseases, are infectious neurodegenera-
were analyzed. In addition, 2 strains of knockout (KO) mice lacking
expression of cytokines of possible importance to disease were in-
MATERIALS AND METHODS
Mice. All mice were housed at the Rocky Mountain Laboratories (RML)
in an AAALAC-accredited facility, and experimentation followed NIH
RML Animal Care and Use Committee-approved protocols. Female
C57BL/10/SnJ mice were used for the initial scrapie pathogenesis experi-
ments (Jackson Laboratories, Bar Harbor, ME). Female B6.129S1-
C57BL/6J (B6) (Jackson Laboratories) mice were used in subsequent
scrapie infection studies. In order to reduce genetic differences in back-
ground genes, KO mice were backcrossed 11 times to C57BL/6 mice be-
fore intercrossing to obtain homozygous KO mice. These three strains
should have nearly identical background genes, and differences among
them are likely due to changes at the targeted KO loci. Mice were pur-
chased as weanlings and were housed in groups of 4 per box.
Inoculation and observation of mice. Mice were anesthetized with
at the age of 4 to 6 weeks with 50 ?l of a 1% (wt/vol) scrapie brain
Received 29 May 2012 Accepted 5 July 2012
Published ahead of print 11 July 2012
Address correspondence to Bruce Chesebro, firstname.lastname@example.org.
*Present address: Déborah Tribouillard-Tanvier, Inserm U1078, Faculté de
Médecine et des Sciences de la Santé, Brest, France.
D.T.-T. and B.R. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
October 2012 Volume 86 Number 19Journal of Virologyp. 10377–10383jvi.asm.org
fetal bovine serum (FBS). C57BL/10/SnJ mice were inoculated with the
mouse-adapted scrapie strain 22L (1.0 ? 10650% lethal doses [LD50]) or
Brains were collected, and the left half was placed in formalin for histologic
analysis, while the right half was flash frozen in liquid nitrogen for later bio-
chemical analysis. Mock-infected age-matched controls were processed in a
with either the 22L (1.0 ? 106LD50), RML (4.0 ? 104LD50), or 79A (1.8 ?
105LD50) strain of rodent-adapted scrapie. Following infection, mice were
itored biweekly, and mice were euthanized when they displayed consistent
signs of ataxia, kyphosis, somnolence, and hind leg weakness. Brains were
Immunohistochemistry. Mice were euthanized; brains were re-
moved; and the left half of each brain was placed in 3.7% phosphate-
buffered formalin for 3 to 5 days before dehydration and embedding in
placed on positively charged glass slides, and dried overnight at 56°C.
Slides were stained with a standard protocol of hematoxylin and eosin
(H&E) for observation of overall pathology. For the detection of PrPres,
pleted in a Discovery XT slide stainer (Ventana, Tucson, AZ).
PrPres was detected as described previously (15). In brief, PrPres an-
tigen was retrieved in a Ventana automated Discovery XT stainer by in-
cubation for 188 min at 95°C in CC1 buffer (Ventana) containing Tris-
borate-EDTA (pH 8.0), followed by (i) staining of PrPres with the
monoclonal human anti-mouse PrP antibody D13 (dilution, 1:500) (In-
ary staining with biotinylated anti-human IgG (dilution, 1:250) (Jackson
ImmunoResearch, West Grove, PA), and (iii) avidin-horseradish peroxi-
Sections were stained for microglia by using the primary antibody
Iba1. Slides were exposed to the standard antigen retrieval procedure (44
min at 100°C) and were stained with anti-Iba1 (polyclonal rabbit anti
Iba1; kindly provided by John Portis) at 1:2,000 for 40 min at 37°C. The
secondary antibody, goat biotinylated anti-rabbit IgG (Biogenex), was
The chromogen used was Fast Red (Red Map kit; Ventana).
Immunostaining of slides for astrocyte detection used a mild anti-
gen retrieval step (22 min at 100°C), followed by (i) staining with
polyclonal rabbit anti-GFAP (Dako) at a 1:3,500 dilution for 16 min,
(ii) staining with goat biotinylated anti-rabbit IgG as a secondary an-
tibody for 16 min, and (iii) streptavidin-alkaline phosphatase using
Fast Red as the chromogen.
All histopathology slides were read either using an Olympus BX51
puter monitors (Spectrum and Imagescope software by Aperio). Images
were obtained using Microsuite FIVE software on the microscope or Im-
agescope software on the computer.
mice. Brains were homogenized (20% [wt/vol]) in PBS containing 0.1
mM Pefabloc, using a Mini Bead Beater (BioSpec Products) as described
previously (35). Brain homogenates were sonicated for 1 min, vortexed
aggressively for 30 s, and frozen in aliquots at ?80°C for future use.
ples were analyzed as described previously (21). Briefly, 20 ?l of a 20%
X-100, and 1% sodium deoxycholate in a total volume of 31 ?l. Samples
were treated with 50 ?g/ml of proteinase K (Roche Diagnostics) for 45
(Roche Diagnostics), and the reaction product was placed on ice for 5
min. An equal volume of 2? Laemmli sample buffer (Bio-Rad, Hercules,
at ?20°C until they were electrophoresed on a 16% Tris-glycine sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Life
Technologies, CA) and blotted to polyvinylidene difluoride (PVDF)
membranes using a 7-min transfer, program 3 (P3), on an iBlot (Life
Technologies, CA) device. Immunoblots were probed with monoclonal
human anti-PrP antibody D13 at a 1:100 dilution (0.66 ?g/ml) using a
supernatant made in our laboratory from CHO cells expressing the D13
peroxidase-conjugated anti-human IgG (dilution, 1:5,000; Sigma, St.
Louis, MO). Protein bands were visualized using an enhanced chemilu-
minescence (ECL) detection system (GE Healthcare).
For PrPres quantification during the course of infection, PrPres levels
measured in brains obtained at the time of clinical disease. All brain ho-
mogenates were treated with proteinase K as described above. Two sam-
brain were tested to facilitate comparisons.
Cytokine quantification. A multiplex assay kit (Bio-Rad, Hercules,
CA) was used to determine the concentration of cytokines in brain ho-
mogenates as described previously (34, 35). Nine cytokines (IL-12p40,
CCL2, CCL3, CCL5, CXCL1, IL-1? IL-9, IL-13, gamma interferon) were
analyzed. Samples were thawed and diluted to the final concentration in
Dulbecco’s modified Eagle medium (DMEM) with 10% FBS. Samples
was analyzed at 4% in order to obtain values in the midrange of the
standard curve for this cytokine. All samples were assayed in duplicate.
Data were obtained using the Bio-Plex Manager software program (ver-
sion 4.1.1; Bio-Rad) for standardization and standard curve acquisition
and were exported to Microsoft Excel (Microsoft Corporation, Seattle,
WA) for further analysis. Statistical analysis was done for each cytokine
using one-way analysis of variance (ANOVA) on GraphPad software to
compare normal control mice with mice for whom data were obtained at
various times after scrapie infection. For these experiments, 3 to 4 unin-
were inoculated with normal brain homogenate material at the same
and 135 days postinoculation (dpi). The results from all these mice ap-
peared to be similar, and all control mice were grouped for comparison
with infected mice for statistical analysis and graphic presentation.
Early PrPres accumulation and gliosis in prion disease. To de-
FIG 1 Early detection of PrPres in scrapie-infected brains. (A) Immunoblot
detection of PrPres in brain at various times after i.c. inoculation with scrapie
strain 22L. The upper panel was exposed for 10 s, and the lower panel was
any bands in the 20-dpi samples. Results similar to those in the lower panel
were also seen when samples were analyzed using the phosphotungstic acid
precipitation method (28). (B) PrPres was quantitated by density scanning of
point is shown as a percentage of the amount detected at the terminal stage.
Tribouillard-Tanvier et al.
jvi.asm.orgJournal of Virology
els of various cytokines in scrapie-infected brain homogenates at
different times after scrapie inoculation. For these experiments,
C57BL/10 mice were infected intracerebrally with 1 ? 10650%
infective doses (ID50) of the 22L strain of mouse scrapie, and
beginning at 20 days postinoculation (dpi). PrPres was first de-
at 20 dpi detected an even distribution of microglia in the same area. (Inset) Thin processes of cells characteristic of nonactivated microglia. (G) At 40 dpi, increased
Cytokines and Scrapie Brain Disease
October 2012 Volume 86 Number 19jvi.asm.org 10379
mulated slowly to about 10% of the terminal level by 100 dpi and
increased rapidly thereafter to reach a maximum at the clinical
stage at around 135 dpi (Fig. 1B).
Infected brain tissue was also analyzed for PrPres by immuno-
histochemistry, because small localized foci of PrPres deposition
might escape detection by immunoblotting of whole-brain ho-
peduncular nuclei (Fig. 2B to D). At 80 dpi (Fig. 2E) and at later
times up to the clinical endpoint (data not shown), PrPres was
liferation and activation of microglia and astroglia is a prominent
feature of prion disease infection in brain.
ing anti-Iba1 and anti-GFAP to detect microglia and astroglia,
respectively. At 20 dpi, Iba1-positive cells had normal-appearing
somata with thin and delicate projecting processes (Fig. 2F) and
were not distinguishable from microglia seen in uninfected mice
glia also appeared similar to those seen in uninfected mice (not
able PrPres, there was notable glial activation. Microglia were hy-
pertrophic with an enlarged, darkened soma and shorter, thicker,
less branched processes (Fig. 2G, H, And I), and astroglia exhib-
ance indicating an activated phenotype (not shown). Gliosis was
more extensive at 80 dpi (Fig. 2J) and increased steadily until the
clinical endpoint at 135 dpi.
Early cytokine expression in prion disease. In our previous
studies, levels of several cytokines were found to be elevated in
more, in vitro cultures of microglia and astroglia stimulated with
scrapie-infected brain homogenates were found to release cyto-
kines. Since gliosis was detected as early as 40 dpi in the brains of
mice infected with scrapie (Fig. 2), we investigated whether cyto-
kines were also elevated in the brain at early times after scrapie
inoculation. Levels of 9 cytokines in brain homogenates from the
analysis revealed a detectable increase above background for sev-
eral cytokines. Relative to expression in control mice, IL-12p40
was first detected in scrapie-infected mice at 60 dpi and showed a
progressive increase throughout the disease, with a 30-fold in-
3A). CCL3, IL-1?, and CXCL1 were also elevated by 80 dpi, and
to F). Thus, there was a reproducible elevation of the levels of
early preclinical phase of scrapie infection. However, it was not
clear whether these cytokines were actively contributing to the
progressive brain damage observed or whether these cytokine re-
sponses were part of the host response attempting to repair the
scrapie-induced brain damage.
Scrapie infection in IL-12p40 and IL-12p35 knockout mice.
detectable brain alterations and also persisted throughout the
course of the disease, we examined whether IL-12p40 was re-
quired for scrapie-induced disease by infection of IL-12p40 KO
mice. Since IL-12p40 forms a heterodimer with IL-12p35 in the
active IL-12 cytokine, we also examined IL-12p35 KO mice (for a
review, see reference 6). C57BL/6 (B6) mice were used as controls
for both types of KO mice.
In these experiments, we tested three different scrapie strains
(22L, RML, and 79A) because of potential strain-specific differ-
ences in the pathogenic processes. In these experiments, each KO
mouse type studied developed terminal scrapie disease at mean
times similar to those for B6 control mice inoculated with the
same scrapie strain (Fig. 4A and B). Disease progressed slightly
this strain was approximately 10-fold higher than those of the
the tempo or incidence of prion-induced disease in mice.
To see whether the disease induced in these KO mice differed
FIG 3 Detection of cytokines in brain tissue at various times after scrapie
infection. Protein levels of 9 cytokines in the brains of scrapie 22L-infected
mice were measured by multiplex assays at various days postinoculation. The
those for normal control mice (N) at various days postinoculation. Data for
CCL2 were obtained from samples assayed at a 4% brain concentration, and
other cytokines were assayed at a 1% brain concentration. IL-9, IL-13, and
gamma interferon were also analyzed, but no values above those for normal
controls were detected. Two to four mice per group were studied. Statistical
analysis was carried out using one-way ANOVA with GraphPad Prism soft-
ware. ***, P ? 0.001; **, P ? 0.01; *, P ? 0.05.
Tribouillard-Tanvier et al.
jvi.asm.org Journal of Virology
from that seen in B6 control mice, we also tested several other
parameters of scrapie disease pathogenesis in these experiments.
mice and B6 mice had similar intensities and similar glycoform
banding patterns for each scrapie strain tested (Fig. 4C and D).
as well as vacuolation of the gray matter by H&E staining, all of
mice (Fig. 5). In addition, at the clinical endpoint after scrapie
infection, cytokine levels in the brain were similar to those re-
ported previously (35) (data not shown). Thus, these results gave
no evidence that lack of expression of IL-12p40 or IL-12p35 al-
tered the basic parameters of brain disease with any of the three
scrapie strains tested.
In the present experiments, we found evidence for PrPres deposi-
tion and both astrogliosis and microgliosis in the brain at 40 days
these findings were detectable. Thus, these three aspects of prion
40 dpi. PrPres detection at 40 dpi in this system was 20 to 30 days
earlier than in several previous studies (1, 2, 4, 12, 17, 30). This
might be due to the rapid tempo of the disease induced by our
stock of strain 22L, which is known to induce clinical disease at
135 to 140 dpi and is about 15 days faster than our stocks of other
scrapie strains, such as RML, ME7, and 79A. This early time of
onset of the pathological process in the brain should provide an
opportunity for future detection of key early events in scrapie
Using quantitative multiplex cytokine protein analyses with
the same group of mice, we found elevated levels of several cyto-
kines, including IL-12p40, CCL2, CCL3, CCL5, IL-1?, and
CXCL1, starting at 60 to 80 dpi. These cytokines were previously
shown to be present in scrapie-infected brains at the end stage of
in vitro stimulation with scrapie-infected brain extracts (35).
These cytokines appeared slightly later than the initial PrPres de-
posits and gliosis, suggesting that cytokine elevation might be in-
duced by these earlier pathological events. Alternatively, our
biochemical cytokine assay based on analysis of whole-brain ho-
mogenates might not be sensitive enough to detect small foci of
early cytokine production simultaneous with, or even preceding,
PrPres deposition and gliosis. Because of these caveats, we cannot
distinguish whether these cytokines contribute to the ongoing
attempting to repair this damage.
The study of mice in which individual cytokine genes have
been deleted is a powerful tool for testing the requirement for
individual genes in disease pathogenesis or recovery. In previous
studies of cytokine or cytokine receptor knockout mice using i.c.
scrapie infection, a variety of outcomes have been seen. Depend-
ing on the individual gene tested and the scrapie strain utilized,
survival times either were unchanged (IL-6 , IL-4 and/or
IL-13 , MyD88 ), were slightly increased (CCL2 with
strain ME7 , CXCR3 , or IL-1R ), or were slightly de-
creased (CCL2 with strain RML/Chandler , IL-10 , or
CCR1 ). Thus, although most of these cytokines, cytokine
neuroinflammation and neurotoxicity after scrapie infection, no
single cytokine or receptor has so far been proven to be required
for scrapie disease.
scrapie strains. Each dot represents one mouse. Horizontal bars show the mean incubation period. Values represent the day postinoculation when the animal was
Cytokines and Scrapie Brain Disease
October 2012 Volume 86 Number 19jvi.asm.org 10381
In the current experiments, we attempted to determine
whether prion disease brain pathogenesis was influenced by dele-
tion of IL-12p40, the cytokine with the earliest onset and highest
fold increase during disease. Knockout mice in which expression
of IL-12p35 was deleted were also studied, because a heterodimer
of IL-12p40 and IL-12p35 polypeptides forms the active IL-12
cytokine. In these experiments, scrapie was inoculated by the i.c.
route in order to focus the study on pathogenesis within the cen-
tral nervous system (CNS) rather than on aspects of extracerebral
infection and neuroinvasion (14, 19). After scrapie infection,
these two KO mouse strains showed no differences in survival
time, PrPres occurrence, astrogliosis, microgliosis, or neutrophil
vacuolation from non-KO C57BL/6 control mice (Fig. 4 and 5).
Thus, none of the three cytokines containing IL-12p40 or IL-
for a review)—were required for these aspects of scrapie patho-
genesis, nor were they required for fatal scrapie disease. Since
many of these cytokines act on T lymphocytes and dendritic
cells not found in the CNS during scrapie disease, it may not be
genes. However, in microglia, IL-12p70, the IL-12p40 mono-
mer, and the IL-12p40 dimer all stimulate mitogen-activated
protein (MAP) kinase and extracellular signal-regulated kinase
(ERK) pathways, as well as inducing the production of tumor
necrosis factor (TNF) and nitric oxide (10, 11). Nevertheless,
based on our current experiments, these effects on microglial
function do not influence scrapie disease tempo or survival
after inoculation of any of the three scrapie strains tested in our
This work was supported by the National Institute of Allergy and Infec-
tious Diseases, Division of Intramural Research.
We thank Kimberly Meade-White for technical assistance with the
mouse experiments, Jeffrey Severson for assistance with animal hus-
bandry, Dan Long, Lori Lubke, and Nancy Kurtz for technical assistance
with immunohistochemistry, and Suzette Priola and Karin Peterson for
critical reading of the manuscript.
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