Aberrant Immune Responses in a Mouse with Behavioral
Yong Heo1., Yubin Zhang2,3., Donghong Gao2, Veronica M. Miller2, David A. Lawrence2,3*
1College of Natural Sciences, Catholic University of Daegu, Kyongsan-si, Republic of Korea, 2Wadsworth Center, New York State Department of Health, Albany, New York,
United States of America, 3University at Albany School of Public Health, Albany, New York, United States of America
BTBR T+tf/J (BTBR) mice have recently been reported to have behaviors that resemble those of autistic individuals, in that
this strain has impairments in social interactions and a restricted repetitive and stereotyped pattern of behaviors. Since
immune responses, including autoimmune responses, are known to affect behavior, and individuals with autism have
aberrant immune activities, we evaluated the immune system of BTBR mice, and compared their immunity and degree of
neuroinflammation with that of C57BL/6 (B6) mice, a highly social control strain, and with F1 offspring. Mice were assessed
at postnatal day (pnd) 21 and after behavioral analysis at pnd70. BTBR mice had significantly higher amounts of serum IgG
and IgE, of IgG anti-brain antibodies (Abs), and of IgG and IgE deposited in the brain, elevated expression of cytokines,
especially IL-33 IL-18, and IL-1b in the brain, and an increased proportion of MHC class II-expressing microglia compared to
B6 mice. The F1 mice had intermediate levels of Abs and cytokines as well as social activity. The high Ab levels of BTBR mice
are in agreement with their increased numbers of CD40hi/I-AhiB cells and IgG-secreting B cells. Upon immunization with
KLH, the BTBR mice produced 2–3 times more anti-KLH Abs than B6 mice. In contrast to humoral immunity, BTBR mice are
significantly more susceptible to listeriosis than B6 or BALB/c mice. The Th2-like immune profile of the BTBR mice and their
constitutive neuroinflammation suggests that an autoimmune profile is implicated in their aberrant behaviors, as has been
suggested for some humans with autism.
Citation: Heo Y, Zhang Y, Gao D, Miller VM, Lawrence DA (2011) Aberrant Immune Responses in a Mouse with Behavioral Disorders. PLoS ONE 6(7): e20912.
Editor: Marian Ludgate, Cardiff University, United Kingdom
Received January 27, 2011; Accepted May 16, 2011; Published July 20, 2011
Copyright: ? 2011 Heo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by New York State Department of Health, National Institutes of Health (ES grant to DAL), and an Autism Research Institute
grant to VMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Development of an appropriate experimental animal model is
critical if we are to extend our knowledge of the underlying causes
of abnormal neurobehaviors. Numerous bidirectional immune
and nervous system interactions are known, but we lack an
understanding of the immunological mechanisms implicated in
neuroendocrine developmental effects such as the behavioral
disorders of autism. Aberrant immunity has been reported to be
associated with autism and autism spectrum disorders (ASDs);
however, there have been no studies mechanistically connecting a
particular immune activity with the aberrant behaviors of
individuals with an ASD [1–4]. For example, serum IgE levels
have been reported to be normal [5,6] or elevated [7,8], and
mastocytosis has been reported to be associated with ASD .
There does seem to be consistency regarding elevated levels of
plasma cytokines and neuroinflammation (elevated levels of
cytokines in the brain) being associated with ASD [10–14].
Among the inbred mouse strains that have been tested for
abnormal behaviors, BTBRT+tf/J (BTBR) mice were among the
most autism-like strains; BTBR mice display both low social
approach and resistance to change in routine with the water maze
assay, which is a behavior consistent with an autism-like
phenotype . BTBR mice also show low reciprocal social
transference for food, high levels of repetitive self-grooming, low
levels of social approach and juvenile play, and an unusual pattern
of ultrasonic vocalizations; these traits respectively reiterate the
impaired communication, repetitive behavior, and lowered
reciprocal social interactions of autistic humans [15–18]. BTBR
mice have severely reduced corpus callosum and hippocampal
commisures , and reduction of corpus callosum and
hippocampal commisures has been observed in some autistic
children [20,21]. Results on neuroanatomical and behavioral
evaluations of BTBR mice are being accumulated, but no reports
are available on the immunological characteristics of these mice. If
cross-talk between the immune system and the nervous system
 and the putative contribution of autoantibody (AutoAb)-
mediated neuroinflammation to pathogenesis of ASDs are
considered together, a systemic assessment of immune functions
in BTBR mice could provide important clues to help to delineate
the involvement of the immune system in the incidence and
progression of ASD.
Immune system activities have been implicated in the
development of ASD as well as in some of the associated
pathophysiology. A broad spectrum of immune abnormalities,
including aberrant mucosal immunity, has been reported for
autistic subjects . Additionally, serum Abs against central
nervous system (CNS) antigens (Ags), and maternal Abs to fetal
brain proteins, have been associated with ASD [23–26]. Maternal
IgG reactive to fetal brain proteins contributing toward ASD
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development has been experimentally shown via induction of
behavioral alterations in offspring mice or monkeys that were
prenatally exposed to serum IgG obtained from mothers of autistic
children [27,28]. Thus, neuroinflammatory processes in the brain
may play a role in the induction of the autistic behavioral changes
induced by Abs.
To determine the relationship between the immunoreactivity
and the abnormal behaviors that have been observed in BTBR
mice, we analyzed various immune aspects of these mice,
including presence of Abs to brain Ags, immune cell distribution
in the periphery and brain, expression of proinflammatory
cytokines in the brain, Ab production to KLH and host resistant
against Listeria monocytogenes (LM, an intracellular pathogen). Not
surprisingly, analyses of F1 offspring from two different crosses
using the BTBR strain have indicated that the offspring have less
anti-brain activity and neuroinflammation than is seen for the
homozygous BTBR strain, but offspring from BTBR dams have
greater anti-brain responses than offspring from B6 dams. The
BTBR strain has elevated B cell activity that appears to have a
predominant Th2-like profile, which may account for the BTBR’s
elevated immunity to brain Ags. Although the maternal environ-
ment has an influence on the immune status of the offspring, the
BTBR genetic influence on behavior is suggested since the F1 mice
of BTBR and B6 matings appear to have normal behavior
although the F1 mice have less sociability than B6 mice.
Enhanced amount of IgG deposited in the brain of BTBR
One of the major characteristics of autoimmune neurologic
disorders is the generation of AutoAbs with specificity for various
components of the nervous system [22,29]. Since various AutoAbs
against central nervous tissue antigens have been reported from
autistic subjects , we queried whether a similar phenomenon
was observable in BTBR mice. Human studies performed for
evaluation of Abs against brain antigens have used serum as a
source of Abs and specific neuronal proteins or else human brain
protein medleys as sources of brain antigens [24,25,28,31–35]. In
our study, we first measured the amounts of IgG deposited in the
whole brain or in separate brain regions of perfused mice; the
presence of IgG was determined by ELISA, and was compared
among the BTBR and B6 strains and their offspring. B6 mice were
chosen as a control strain since, unlike BTBR mice, B6 mice
demonstrate normal social behaviors and B6 mice have previously
been used for the comparative behavioral analyses [15,19,36];
additionally, B6 mice are considered immunologically normal, and
they have the same major histocompatibility complex (H2b) as
BTBR mice. The IgG levels in whole-brain homogenates of
BTBR mice (male: 98.4621.3, female: 104.8627.4 ng/mg
protein) were approximately 2-fold higher than those of the F1
offspring (BCF1 male: 40.262.8, BCF1 female: 48.2611.0, CBF1
male: 48.067.4, CBF1 female: 44.565.9 ng/mg protein), and 4-
fold higher than those of the B6 control mice (male: 21.164.4,
female: 13.663.0 ng/mg protein) (Fig. 1). There was no sexual
dimorphism, but there was a significant strain difference
(F=11.45, DF=(3, 20), p,0.001); post-hoc analysis indicates
BTBR brains contained significantly (p,0.001) more IgG than
did B6 brains, but there were no differences between the F1
offspring and between F1 offspring and B6 mice.
Although there was no gender difference within a strain with
regard to IgG deposited in the brains (Fig. 1), we still separately
evaluated males and females for the regions with deposited IgG.
Again, there were no significant gender differences; therefore, the
results of males and females of each strain were pooled (Table 1).
Based on two-way ANOVA, the only brain regions that
significantly differed were substantia nigra (SN) and hippocampus
(HC); additionally, regional amounts of IgG in BTBR mice
differed from that of all of the other strains, and BCF1 results
differed from those of B6. Overall, the SN was the region that
showed the greatest accumulation of deposited IgG, and the
striatum (STR) or HC had the lowest amounts of IgG.. The
increased amount of IgG in the brains of the BTBR mice
compared to those of B6 mice could not be further delineated by
immunohistochemistry, in that perfused non-fixed brain sections
from BTBR and B6 mice that were washed in vitro with PBS
demonstrated no obvious differences in IgG distribution (data not
Based on the time spent in the chamber with the novel mouse vs.
the time in the chamber with the empty cage, adult ($pnd70)
BTBR mice did have less sociability than the B6, CBF1, and BCF1
strains (Fig. 2). This is consistent with the previous evaluation of
BTBR and B6 mice by this behavior assay [15,17]. There were no
significant differences for gender. Although unlike the BTBR mice,
the F1 strains spent more time with the novel mouse than with the
novel object (Fig. 2A), they did display less sociability than the B6
mice (Fig. 2B). Thus, the intermediate behaviors of the F1 mice
appear to reflect the intermediate amounts of IgG in the brains of
the F1 mice (Fig. 1). Kruskal-Wallis one-way ANOVA indicated
significant strain differences, and Dunn’s pairwise analysis
indicated that BTBR mice had less sociability than the CBF1
and B6 mice.
Upregulation of IgG and brain-reactive IgG expression in
the peripheral blood of BTBR mice
Since there was significantly more IgG present in the perfused
brains of BTBR mice than in those of the F1 offspring or B6 mice,
we measured the amounts of total serum IgG (Fig. 3) and the levels
of serum IgG binding to brain homogenates (Fig. 4) of pnd21
BTBR, BCF1, CBF1, and B6 mice. The level of total IgG was
highest in BTBR mice, lowest in B6 mice, and intermediate in
BCF1 and CBF1 mice. Two-way ANOVA indicated no
interaction or gender significance, but there was strain significance
(F=20.91, DF=(3, 80), p,0.0001). One-way ANOVA (Kruskal-
Figure 1. BTBR mice have an enhanced amount of IgG in their
perfused brains. Whole brain homogenates were obtained from 4
BTBR males or females, 3 BCF1 males or females, 4 CBF1 males or
females, and 5 B6 males or 4 females at postnatal day 21. Mice were
perfused with PBS prior to the brain collection. *, p,0.05 vs. B6 males;
**, p,0.01 vs. B6 females.
Immunity of BTBR T+tf/J Mice
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Wallis test) based on strain indicated p,0.0001 and Dunn’s post-
hoc test indicated BTBR mice had higher total IgG levels
(p,0.001) than did CBF1 and B6 mice, and BCF1 mice had
higher total IgG levels (p,0.001) than did B6 mice. Based on a
comparative study of B6 mice with five other strains, the IgG levels
of BTBR or BCF1 pups are not higher than those of B6 pups
because of abnormally low IgG levels in B6 mice since B6 mice
have relatively high levels of serum IgG (http://phenome.jax.org)
and of peripheral blood B cells .
The presence and amounts of brain-reactive IgG in serum
samples from BTBR mice were compared with those assessed for
BCF1, CBF1, and B6 mice. Homogenates from whole brain or
from each of six different brain regions from SCID mice were used
as Ags to detect brain-reactive IgG through ELISA. As for total
serum IgG levels, the anti-brain Ab levels in sera were not different
by gender, but they were significantly different based on strain
(F=20.99, DF=(3, 37), p,0.0001). The level of brain-reactive
IgG against whole brain homogenates was higher in BTBR mice
than in CBF1 or B6 mice (p,0.001), and male BCF1 anti-brain
levels were greater (p,0.05) than those of B6 mice (Fig. 4).
Analysis (three-way ANOVA for sex, strain and brain region) of
serum IgG binding to various brain regions (cortex, CTX; STR;
HC; hypothalamus, HT; SN; cerebellum, CB) showed overall
significance for strains (F=118.502, DF=3, p,0.001) that the
brain-reactive IgG levels were highest in the brain regions tested
with sera from BTBR mice; intermediate binding activity was
obtained with sera from BCF1 mice, and sera from B6 mice
showed the lowest binding; there were no differences between
CBF1 and B6 mice (Table 2). Additionally, there were differences
in the amount of IgG binding to SN vs. all other regions and HT
vs. HC and CB. As shown for whole brain homogenate (Fig. 4),
Table 1. Levels of IgG present in brain regions.a
IgG level (ng/mg protein)b
BTBR151641103626 94617 110624211635159636
BCF16269 666125865 7661210962574610
CBF14262 4064 3564 5463 566359611
B62367 2067 2266 2364 2767 2968
aEach brain region was isolated from perfused postnatal day 21 mice, and the homogenates were used for evaluation of IgG level deposited in each region. Number of
mice was 8 for BTBR, and 6 for the other strains. The results are expressed as mean 6 SEM. The IgG level in each brain region was significantly different among the
bIgG levels in brain region of the mouse strains were significantly different by two-way ANOVA with BTBR vs. other strains and BCF1 vs. B6; SN also differed from HP.
cpost-hoc test indicates that the regional IgG level of BTBR differs from that of B6 and CBF1.
dpost-hoc test indicates that the regional IgG level of BTBR differs from that of B6.
epost-hoc test indicates that the regional IgG level of BTBR differs from that of B6 and CBF1, and BCF1 differs from that of B6.
fpost-hoc test indicates that the regional IgG level of BTBR differs from that of B6, and BCF1 also differs from B6.
Figure 2. BTBR mice lack sociability. After a 10 min period for the
test mouse to become acquainted with the three chamber box, the test
mouse was assessed (10 min) for the amount of time spent in the side
chamber with the caged novel (BALB/cByJ) mouse versus the opposite
side chamber with an empty cage. C57BL/6 (B6), CBF1, and BCF1 mice
spent significantly (*) more time in the chamber with the novel mouse
(A); however, based on the behavior index (time with mouse minus
time with object), the behavior of the BTBR mice was significantly
different (**) from that of the CBF1 or B6 mice (B).
Figure 3. BTBR and BCF1 mice have significantly higher serum
IgG levels than those of CBF1 or C57BL/6 (B6) mice. Sera were
obtained from postnatal day 21 mice. The numbers of mice were 14 for
BTBR male, 17 for BTBR female, 9 each for BCF1 and CBF1 male or
female mice, and 11 for each B6 male or female. *, p,0.001 vs. CBF1
and B6 males or females; **, p,0.05 vs. B6 males; and ***, p,0.01 vs. B6
Immunity of BTBR T+tf/J Mice
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there were no significant differences between sexes within a strain;
however, BCF1 males exhibited significantly higher amounts of
brain-reactive IgG against CTX, SN, and CB homogenates than
did B6 males, and no corresponding finding was observed for the
females of these two strains. These observations suggest that the
antigenic targets of the elevated levels of the IgG to brain Ags are
not focused in any specific brain regions, but instead are reactive
to Ag(s) common throughout the brain regions; however, the
regional differences may relate to the proportion of different cell
types in each region or relative accessibility of serum constituents.
When all of our IgG results are considered together, a consistent
upregulation of the levels of IgG was apparent in the sera and
brains of BTBR mice, relative to what was seen for the other
strains; also, BCF1 offspring (BTBR dams and B6 sires), especially
males, showed higher IgG levels than did B6 mice. However, the
IgG levels did not differ between CBF1 and B6 mice. A high
constitutive polyclonal B cell activation in the BTBR mice could
lead to high endogenous systemic inflammation.
Blood brain barrier (BBB) integrity
BBB leakage or increased BBB permeability can cause
extravasation of virus, immunoglobulin, or toxic molecules into
brain tissues [38–40]. To investigate whether increased BBB
permeability was a factor contributing to the increased amount of
deposited IgG in the brain of the BTBR mice, we assessed leakage
of Evans blue (EB) dye into the brain after intraperitoneal injection
of the dye (Fig. 5). The BBB permeability index, which reflects the
amount of EB dye per unit weight of brain relative to the amount
of dye per unit volume of plasma, did not differ between BTBR
and B6 mice.
Immune cell subpopulations in the peripheral organs
and brain of BTBR mice
The various types and proportions of immune cells distributed
throughout the peripheral immune organs and brain have not
been previously evaluated for BTBR mice. Since some immune
cell types in the periphery or CNS, such as microglial cells or
CD4+T cells, have been suggested to be associated with
pathogenesis of neuroinflammatory disorders, including autism
[23,41–44], we evaluated proportions and actual numbers of
major immune cell types in the spleen, mesenteric lymph nodes,
peripheral blood, and brain of BTBR mice, and compared the
levels with those for B6 mice (Tables 3 & 4, Fig. 6). Given our
described result showing relatively high levels of IgG in BTBR
mice, we also enumerated plasma cells. The number of CD8+T
cells was significantly greater in the mesenteric lymph nodes of the
BTBR mice than those of B6 mice, but this difference was not
obtained for analysis of spleens or blood (Table 3). Although the
serum IgG levels were elevated in BTBR mice compared with the
IgG levels of B6 mice, the pnd21 BTBR mice did not have any
significant differences from B6 mice with regard to the number of
B cells or plasma cells in the spleen, lymph node or blood (Table 3).
BTBR and B6 mice (pnd60) also were assayed for splenic B cell
numbers and the levels were still equivalent (BTBR, 38.362.6 and
B6, 37.462.76106/spleen). A subset of mice were assayed for B1
Figure 4. BTBR and BCF1 mice have significantly higher
amounts of serum anti-brain antibodies than CBF1 or C57BL/
6J (B6) mice. Sera from postnatal day 21 mice were added to wells
coated with BALB/c SCID whole brain proteins (10 mg/well). The
numbers of sera were 6 for BTBR male or female, 6 for BCF1 male, 3
for BCF1 female, 5 for CBF1 male or female, and 7 for B6 male or female.
The level of brain-reactive IgG was determined by using the HRP-
conjugated goat anti-mouse IgG. *, p,0.01 vs. B6 males or females, and
**, p,0.05 vs. B6 males.
Table 2. Serum IgG binding to Ags of various brain regions.a
Level of brain-reactive IgG (optical density)b
Strain SexCortex (CTX) Striatum (STR) Hippocampus (HC)Hypothalamus (HT)c
BCF1 Male 0.3860.04 0.3960.04 0.4060.040.3660.030.3360.030.3460.03
Female 0.3160.030.3260.020.3460.02 0.3060.020.2860.01 0.2960.02
CBF1 Male0.2760.020.2960.03 0.3160.030.2760.03 0.2360.020.2460.02
aSera from postnatal day 21 mice were added to wells coated with each brain region protein (10 mg/well). Optical densities were measured as described in Figure 2. The
results are expressed as mean 6 SEM.
bSignificant differences were assayed by three-way ANOVA (sex, strain, region) followed by post hoc tests. There were no sex differences within a strain. Differences (p
value) were as follows: BTBR vs. C57BL/6J (B6) (0.009), CBF1 (0.01), and BCF1 (0.013); BCF1 vs B6 (0.017) and CBF1 (0.025).
cSignificant difference in HT vs. CB (0.005) and HC (0.006).
dSignificant difference in SN vs. CTX (0.004), STR (0.004), HC (0.004 ), HT (0.005), and CB (0.003).
Immunity of BTBR T+tf/J Mice
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(CD19+/CD5+) cells since B1 cells are often implicated in
autoimmune processes. The percentages of splenic or blood B1
cells were not different between BTBR and B6 mice (2.960.8% vs.
3.260.7% for spleen and 0.460.2% vs. 0.560.2% for blood). The
percentage of plasma cells (CD45+/CD138+) cells in the bone
marrow also was not significantly higher in the BTBR mice
(BTBR, 4.260.6 and B6, 2.960.4%). The BTBR mice did have a
greater number of CD4+T cells in the mesenteric lymph nodes
(Fig. 6). CD4+T cell differences in both the proportions in
peripheral blood (BTBRmale:
28.463.0, B6 male: 17.061.9, B6 female: 19.161.0%) and the
actual numbers in mesenteric lymph nodes (BTBR male: 8.261.6,
3.260.36106cells) attained statistical significance.
Figure 6. Elevated number of CD4+T cells in various peripheral
organs of BTBR mice, compared with those of B6 mice. Spleens,
mesenteric lymph nodes, and peripheral blood were obtained from
postnatal day 21 male or female mice (n=3 for each sex per strain).
Since there were no gender differences within a strain, the results were
pooled by strain. Percentages (A) or absolute numbers (B) of CD4+T
cells among CD45+cells were determined by flow cytometric analysis.
Number of cells indicates the number per spleen, three pooled lymph
nodes, or 1 ml of blood. *, BTBR vs. C57BL/6 (p,0.05).
Figure 5. No difference in BBB permeability in BTBR mice
relative to B6 mice. Evans blue solution (50 mg/g body weight) was
injected into the peritoneum; 5 h later, peripheral blood was obtained
through cardiac puncture. Brains from the perfused mice were
incubated in formamide solution for 72 h for extraction of the dye,
and the supernatants were collected through centrifugation. Optical
density of plasma or brain supernatant sample was measured at
620 nm. No statistical significance was observed between BTBR and
C57BL/6J mice. Brains were removed after PBS perfusion and weighed.
Table 3. Distribution of lymphoid cell subpopulations in the
peripheral immune organs.
Organ StrainCD8+T cell CD19+B cell
C57BL/6J 11.260.541.961.9 0.3860.06
Lymph nodeBTBR 20.462.027.062.1 0.9360.43
Peripheral blood BTBR13.461.0 48.062.8c
SpleenBTBR 3.160.8 9.561.2190.1669.2
Lymph nodeBTBR 3.360.2c
C57BL/6J 2.160.23.060.4 173.9656.2
Peripheral blood BTBR1.5560.485.4761.000
aPeripheral immune organs were obtained as described in FIGURE 5. The results
are expressed as mean values 6 SE of (a) percentages of gated CD45+cells or
(b) number of cells (blood6103/ml; organ6106for CD8+T cells and B cells and
6103for CD138+plasma cells). Results are for whole spleen, lymph nodes -
three mesenteric lymph nodes, and 1 ml of blood.
cSignificant difference (p,0.001) between BTBR and C57BL/6J; normality
dSignificant difference (p,0.002) between BTBR and C57BL/6J by Mann-
Table 4. Proportions (%) of brain immune cell components.a
Microglial cell Dendritic cell Mast cell
CBF1 88.862.8 1.460.40.860.2
aBrains were collected after perfusion from postnatal day 21 mice.
Flow cytometric analysis was applied to determine proportions of brain
immune cell components among CD45+cells. The results are expressed as
mean values 6 SEM.
bsignificantly different from that of B6 mice.
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The types of immune cells (CD45+hematopoietic cells) in the
brain of BTBR, BCF, CBF, and B6 mice also were assayed
(Table 4). As noted earlier, neuroinflammation could be a factor
associated with the incidence of autism; thus, we assessed the
percentage of brain immune cells known to be related to
inflammation and pathogenesis, such as microglial cells, dendritic
cells, and mast cells [41,42,45]. Two-way ANOVA (sex, strain)
indicated a strain difference (F=4.19, DF=3, p,0.017) followed
by one-way ANOVA (strain, p,0.014) and SNK post-hoc test,
which indicated that the percentage of microglia in BTBR brains
was different from that of CBF1 and B6 mice. Although the
numbers of dendritic cells and FceR1 expressing cells (presumably
mast cells) in the brains of the BTBR mice were slightly greater
than those of CBF1 and B6 mice, the differences were not
significant by flow cytometric analysis, but the microglial cell
percentage for BCF1 mice was significantly (p,0.05) higher than
that for CBF1 or B6 males.
Mast cells were more prominent in BTBR mice
Mast cells are rarely seen in healthy brains but have been
reported to be sparsely present in the thalamus, hypothalamus,
circumventricular organs, meninges and cerebral cortex of rodent
brains . We were unable to characterize a significant increase
in the number of mast cells from cell suspensions of perfused
BTBR brains by flow cytometry, we did observe a significant
increase of mast cells. However, when forebrain, midbrain and
cerebellar tissues encompassing Bregma +1.4 mm to Bregma
26.12 mm from male and female BTBR and B6 mice were
stained with acidic toluidine blue, metachromatic mast cells were
visible in the tissue from BTBR mice at the hippocampal fissure,
lateral thalamic nuclei and third ventricle, but not at the caudate
nucleus, cortex or cerebellum (Figure 7). The mast cells were
perivascular and appeared to be entering brain parenchyma.
There was a noticeable absence of mast cells in all of the
aforementioned brain regions of the male and female B6 mice.
Additionally, we noted increased numbers of mast cells in
meningeal tissues from the BTBR mice.
BTBR mice have increased numbers of IgG-secreting B
cells but not increased numbers of total B cells
Although there were slightly lower numbers of B cells in BTBR
mice, ELISPOT analysis indicated that there are more IgG-
secreting B cells in BTBR spleens (239647.9/105cells; N=13)
than in B6 spleens (60654/105cells; N=14).
Expression of proinflammatory cytokines in brains of
Despite a lack of consensus, a role of cytokine involvement,
neuroinflammation, and/or skewed helper T cell reactivity has
been suggested in the pathogenesis of autism [23,41,47,48]. In
addition, some cytokines have been reported to influence brain
development, and abnormal expression of these cytokines has been
considered to contribute to behavioral aberration in autistic
subjects [23,43,49]. Therefore, we examined the expression of
various cytokines in the whole brain (Fig. 8) and in individual brain
regions (Table 5) of BTBR, B6, and F1 mice, focusing on
proinflammatory cytokines such as IL-33, IL-18, IL-1b, IL-6, and
TNFa. Since there were no gender differences for IgG levels, the
results for males and females were pooled for the cytokines in the
whole brain homogenates (Fig. 8). First, in terms of the level of
cytokine expression in the whole brain, most of the statistically
significant differences were found to exist between BTBR and B6
mice; no apparent difference was observed between BCF1 and
CBF1 mice. Expression of three of the representative proin-
flammatory cytokines (IL-33, IL-18, and IL-1b) was higher in the
BTBR mice than in B6 mice, and appeared intermediate in the
BCF1 and CBF1 mice; the expression of IFNc and of TNFa was
low in BTBR brains (data not shown). The levels of IL-6 and IL-10
also were higher in the BTBR mice (0.4660.07 for IL-6 and
0.7760.40 for IL-10 pg/mg protein) than in the B6 mice
(0.2460.04, IL-6; 0.2560.07, IL-10 pg/mg protein), but the
differences were not significant. The expression levels of IL-12 or
IL-2 differed little across the strains, except that the IL-2 level was
lower BCF1 females (data not shown). Overall, IL-33, IL-18, and
IL-1b were expressed to a greater extent in the whole brain of
BTBR mice than in the whole brain of B6 mice.
The pattern of cytokine expression in each brain region (Table 5)
was similar to the pattern seen for the whole brain (Fig. 8). In
general, the expression levels of cytokines were highest in BTBR
mice and lowest in B6 mice. Interestingly, there were significant
strain differences for IL-33 in all brain regions; this was true only
for IL-33. Surprisingly, the region with the most significant strain
differences was the substantia nigra (SN); the SN is a very small
brain region in which microglial cells and dopaminergic neurons
are of higher proportion than most other brain regions . In
general, the SN and the cerebellum had the greatest differences
between BTBR and B6 mice for the most cytokines. Notably,
there were significantly lower levels of IFNc and TNFa in whole
brains of BTBR mice, in comparison with B6 whole brains, but
this was not reproduced when the individual brain regions were
Type-1/type-2 immune balance
Since the BTBR mice expressed high levels of IgG and
cytokines, we investigated humoral immunity to KLH and innate
and cell-mediated immunity to LM of adult (pnd70) BTBR and B6
mice. To eliminate differences due to estrous cycle, only males
were assessed. Primary and secondary anti-KLH levels were
significantly higher in BTBR mice than the levels of B6 mice
(Fig. 9). Immune defenses against LM were opposite of the BTBR
and B6 responses observed for humoral immunity to KLH; BTBR
mice had significantly less immunity and thus the cfu levels of LM
were greater in livers and spleens. For BTBR mice, there were
2.160.361010cfu LM in the liver and 4.660.56108cfu LM in
the spleen, whereasB6 mice
0.760.26106cfu LM, respectively (Fig. 10). In previous studies
with BALB/c mice, which are more susceptible than B6 mice to
listeriosis, there were always lower cfu values than observed with
BTBR mice. Since, innate and Th1 immune responses are
responsible for defenses against LM, it seems that BTBR mice
have poor innate and type-1 immunity. Interestingly, at 3 days
after infection, the liver levels of IL-17, TGFb, and IL-10 were
significantly greater in BTBR mice than B6 mice (data not shown).
The anti-KLH and LM immune responses suggest that BTBR
mice have predominantly type-2 immunity. This suggestion is
supported by the constitutively high levels of IgE in the sera of
BTBR mice, which were significantly greater than those of B6
mice or the F1 offspring (15.461.3, BTBR; 1.660.2, B6; 3.460.7,
CBF1; 2.860.3, BCF1; mean IgE mg/mL 6 SEM). These results
included pooled sera from males and females; however, most
BTBR females had higher IgE levels than BTBR males (males,
11.961.5; females, 19.462.0). Since the serum IgE levels of the
BTBR mice were elevated to an even greater extent than that of
the IgG differences of BTBR and B6 mice, we assess, whether like
IgG, IgE was elevated in the brains of the BTBR mice.
Additionally, mast cells have been implicated in autism  and
in the enhancement of immune cell entrance into the brain ,
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and mast cells are triggered by IgE to release neurotransmitters
and cytokines. Additionally, the BTBR mice do have more mast
cells surrounding and in the brain. Thus, measurement of IgE
presence in brain regions is of potential relevance to the
heightened neuroinflammation of BTBR mice. Interestingly, the
regional proportions of IgE (Table 6) were similar to those of IgG
(Table 1) in the brains of the BTBR mice; however, the differences
regarding the amounts of IgG and IgE in the brain regions of
BTBR and B6 mice were not similar. All brain regions of BTBR
mice had more IgG than those of B6 mice, but only the CTX,
FCTX and HC of BTBR mice had more IgE. It is also interesting
to note that the higher serum levels of IgE in BTBR females
compared to BTBR males is observed as higher levels only in the
STR, SN, HC, and CB.
In the present study, we have evaluated the basic immunological
characteristics of BTBR mice. The BTBR strain has been reported
to have abnormal behaviors that resemble autism [15–19], and
our behavioral analysis supports the BTBR strain’s previously
described lack of sociability. The lack of sociability displayed by
male and female BTBR mice was lost with the F1 offspring;
however, the heterzygosity of the F1 strains results in an
intermediate behavioral phenotype. The F1 offspring spent less
time with the novel mouse and more time with the novel object
than B6 mice. The intermediate behavioral phenotype is apparent
with the behavior index (time with novel mouse minus time with
novel object). With regard to the differential strain phenotypes,
this time differential (behavior index) correlated with a number of
immune parameters, especially IgG deposited in the brain and the
amount of cytokines (e.g., IL-33) present in the brain (Fig. 11).
Thus, the elevated IgG, which includes Abs deposited in the brain,
and neuroinflammation inversely correlates with degree of
sociablity. Functional alterations of the peripheral or central
immune system have been studied in some individuals with ASD,
and the implication of brain inflammation or generation of
AutoAbs against brain antigens has been discussed, in the context
of the background mechanisms of autism development [23,29].
Beyond the documented autism-like behavior of BTBR mice [15–
17,26,53,54], our present investigation demonstrates that certain
immunologic characteristics of BTBR mice compare well with the
Figure 7. Mast cells in BTBR tissues. Diagram shows the level at which mast cells were apparent in the BTBR male and female brains
(approximately Bregma 22.54 mm). Metachromatic staining of mast cells (purple) was clear on the orthochromatic (blue) background in tissue
sections. Mast cells were visible at perivascular spaces in the lateral posterior thalamic nucleus, at the hippocampal fissure, and at the third ventricle
adjacent to choroid plexus tissues, in BTBR male and female mice. Bar is 20 mm. Mast cells were not visible at the aforementioned regions in either
male or female B6 mice, an example of hippocampal fissure tissues underneath dentate granule cells is shown from male and female cells. Only the
orthochromatic (Blue) staining is visible. Bar is 60 mm.
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immune modulations reported from autistic subjects. Unlike B6
mice, an inbred strain with highly social behavior, 3 week old
BTBR mice exhibited signs of neuroinflammation. BTBR mice
expressed higher levels of brain-deposited IgG, and IgE, brain
cytokines, and MHC class II expressing microglia than these levels
in B6 mice; the peripheral level of CD4+T cells also was higher in
BTBR mice. The neuroinflammation could be due to the activated
microglia or increased presence of mast cells, which were
prominent within the meninges and brains of BTBR mice,
especially at circumventricular organs, particularly the IIIrd
ventricle, the hippocampal fissure, and perivascular spaces in the
posterior lateral thalamus; these areas may play a role in the
BTBR behavioral abnormalities.
BTBR mice also appear to have a predominant Th2 profile, in
that they have high humoral immune responses to KLH, low
immunity to LM, and significantly higher levels of serum IgE. If all
of our data are taken together, BTBR inbred mice appear to be a
strain highly susceptible to neuropathology, due to the elevated
levels of cells, antibodies, and cytokines in their brains. Most
interestingly, the two types of offspring from F1 crosses, BCF1 and
CBF1 mice, have phenotypes intermediate between those of the
parental BTBR and B6 mice, although the BCF1 mice seem to
show slightly increased signs of neuroinflammation than do the
CBF1 mice. These immunologic differences suggest that the
BTBR strain could be a useful model to further study the
developmental influences of immunity on aberrant behaviors.
There is overall consensus on male predominance for autism
prevalence [55,56]. Even though various genetic variations in X
chromosome of autistic subjects have been reported, the
relationship of such variance with pathogenesis of autism or
ASD, especially with regard to a greater percentage of males
having ASD (4:1, male:female, ratio), remains in doubt [57,58].
Likewise, two quantitative trait loci found in X chromosome of
BTBR strain were suggested to modulate the corpus callosum
abnormalities associated with autism , but these chromosomal
variations did not give rise to a gender difference in the anatomical
brain defects . Among autistic subjects, no striking difference
between genders has been reported for immune abnormalities
[23,29]. Interestingly, for monozygotic male and female twins,
concordance was reported to be 86 and 100%, respectively, for an
ASD outcome, and for dizygotic male-male and female-male
twins, the respective concordance values were 40% and 20% .
Thus, yet undefined gender influences appear to exist. Gender
influences were not evident in our analysis of the central and
peripheral levels of IgG in BTBR mice; there also were no gender
differences with regard to expression of proinflammatory cytokines
and immune cell composition. Most published studies of the
autism-like behaviors of BTBR mice have employed male mice
only; no published data have indicated a significant behavioral
difference between male and female BTBR mice. Therefore, the
current assumption seems to be that sex-linked genetic differences
do not influence the development of proinflammatory and
autoimmune-prone immunologic characteristics of BTBR mice.
Nevertheless, some genetic contribution, especially maternal
nuclear or mitochondrial genes likely contribute to the immune
modulations, given that BCF1 mice born of BTBR dams showed
higher autoreactive IgG level, microglial cell percentage, and
expression of proinflammatory cytokines than did B6 mice.
Genetically, the two F1 strains differ in terms of mitochondrial
inheritance as well as X- and Y-linked genes and potentially early
maternal epigenetic influences.
With respect to maternal influence on the autistic phenotype of
offspring, two published experimental approaches provide a hint
as to the role of maternal IgG against fetal brain antigens, in the
induction of behavioral alterations after birth [27,28]. Both of the
studies used serum IgG purified from the blood of mothers of
autistic children, which was injected intravenously into pregnant
Rhesus monkeys or was injected intraperitoneally into pregnant
Figure 8. Brain expression of cytokines. Whole-brain homogenates were obtained from postnatal day 21 mice, as described. IL-18 and IL-33
were assayed by ELISA and all of the other cytokines were assayed by Luminex, including those not detected and therefore not shown. IL-33, IL-18,
and IL-1b were significant based on one-way ANOVA on Ranks with p=0.019, 0.007, and 0.013, respectively (*).
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B6 mice. Reactivity of serum IgG against fetal brain proteins was
confirmed through Western blot analysis or immunohistochemical
staining. Rhesus monkey infant offspring and adolescent/adult B6
offspring that had been prenatally exposed to IgG from mothers of
autistic children displayed higher levels of stereotypic and
hyperactive motor activity than did controls ; the prenatally
exposed mice, as adults, displayed less social interactions with
strangers than did controls . The two studies provided clear
evidence for involvement of antibody in the development of
autistic behaviors in offspring. Our present findings indicate that
the level of anti-brain IgG is higher in BTBR or BCF1 mice born
of BTBR dams than in B6 or CBF1 mice born of B6 dams.
Analyses in the literature of serum from autistic children or
mothers of autistic children have detected Abs against various
CNS Ags, including myelin basic protein, serotonin receptor, glial
fibrillary acidic protein, brain-derived neurotrophic factor, and
heat shock proteins [24–26,33,34]. A role of AutoAbs in the
pathogenesis of autism is not proven, nor is the involvement of
autoimmune mechanisms in ASD. However, clearly, identifying
the specificities and etiologic roles of AutoAbs should be further
Cytokines affect the development of neuronal or glial cells, as
well as behavioral phenotypes [43,61]. In autistic subjects,
immune alterations, particularly cytokine modulations, that affect
the pathogenesis of autism development, are not agreed upon
[23,62]. Vargas et al.  provided evidence for an ongoing
process of neuroinflammation in the CNS of their autistic subjects.
Macrophage chemoattractant protein-1, a proinflammatory cyto-
kine, was the typical cytokine prominently expressed in both brain
and cerebrospinal fluid of autistic patients. Marked activation of
microglial cells and astrocytes was also evident across the brain
regions, especially in the cerebellum. An anti-inflammatory
cytokine, tumor growth factor-b1, was simultaneously elevated in
brain regions indicative of chronic neuroinflammation. In addition
to the usual analysis of IL-1b and TNFa, IL-33 and IL-18 were
assayed in our study. Overall, levels of several cytokines, including
IL-33, IL-18, IL-1b, and IL-6, were elevated, whereas IFNc and
TNFa were decreased in the brains of PND21 BTBR mice,
compared to brains of B6 or CBF1 pups. IL-33, IL-18, and IL-1b
share many traits: synthesis as pro-forms lacking activity,
production of mature/active forms through caspase-1 cleavage,
secretion via endoplasmic reticulum/Golgi-independent pathway,
and biological activity for inflammation [63–66]. Glial cells are a
Table 5. Levels of cytokines expressed in six brain regions.a
Cytokine (pg/mg protein)
Brain region StrainIL-33IL-18 IL-1b
IL-12 IL-2 TNFa
BCF11.360.421.5610.94.061.7 0.260.11.260.92.960.427.465.3 0.660.20.560.1
CBF1 1.760.8 14.365.33.861.20.560.11.360.32.660.533.7613.5 0.560.2 0.460.1
11.366.71.960.90.460.10.660.210.262.1 19.864.0 0.960.1 1.260.2
BCF12.361.113.368.26.963.3 0.660.21.560.5 3.560.749.4613.3 1.318.104.22.168
CBF1 2.761.218.867.6 7.961.80.660.2 1.960.63.960.858.9623.4 1.060.40.760.2
25.7614.87.063.61.460.5 0.760.4 3.861.481.8612.8 3.060.74.461.1
Substantia nigra BTBR38.866.9b
CBF14.062.113.365.5 8.222.214.171.124 1.460.4 3.561.044.6624.00.860.3 0.760.1
aEach brain region was isolated from perfused postnatal day 21 mice as described in Table II.
The results are expressed as mean 6 SEM.
bCytokine levels in brain region of the mouse strains were significantly different by one-way ANOVA.
cpost-hoc test indicates that the regional cytokine level of B6 differs from that of BTBR.
dpost-hoc test indicates that the regional cytokine level of BCF1 differs from that of BTBR.
epost-hoc test indicates that the regional cytokine level of CBF1 differs from that of BTBR.
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major source of IL-33, IL-18, and IL-1b production in the brain.
The percentage of microglial cells was higher in the BTBR mice
than in B6 mice, perhaps related to the higher levels of these
cytokines in the brains of the former strain. Furthermore, when we
compared the mean fluorescence intensity of MHC-II expression
on brain microglial cells, the expression intensity was higher
Figure 9. BTBR males have elevated anti-KLH serum levels. Primary (A) and secondary (B) serum Ab isotypes to KLH were measured by ELISA
for the various strains. The primary and secondary responses were measured 7 days after immunization with 100 mg KLH without adjuvant; * indicates
significant difference from that of the B6 level.
Figure 10. Listeria monocytogenes (LM) burden of infected mice. BTBR and B6 mice were assessed for LM (cfu/organ) in liver and spleen at
threes days after infection; * indicates a significant difference between the two strains.
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(although the difference was not significant) in the BTBR
(6706204) than in the B6 mice (4986314). The greater MHC-
II expression, a phenomenon also observed in Parkinson’s disease
, suggests an elevated state of activation of microglia in the
BTBR mice. Among the above three proinflammatory cytokines,
IL-1b is the only one to have been reported in previous
investigations of immune alterations in autistic subjects [23,62];
the level of IL-1b from stimulated or unstimulated peripheral
mononuclear cells in vitro was higher in autistic children than in
controls. Elevation of IL-18 has been reported for rodents and
humans with psychiatric disorders . Among the brain regions
that we examined, the cerebellum was one that demonstrated
significantly higher expression of both IL-33 and IL-18 in BTBR
mice than in B6 mice; the cerebellum could thus be a ‘‘hot’’ region
for neuroinflammation in the BTBR mice and for ASD in
humans. Loss of Purkinjie cells has been frequently observed in the
cerebellum, and neuronal degeneration and glial cell activation
have predominantly been found in the cerebellum of autistic
The Th1-Th2 paradigm, that is immune skewing toward type-1
cell-mediated immunity (interferon-gamma promoted immunity)
vs. type-2 humoral immunity (IL-4, IL-5 and IL-13 promoted
immunity), may not be properly applicable to immune alterations
in autism. Conflicting results have been derived from human
studies on a Th1 or Th2 shift: several investigations reported Th1
skewedness, but other studies suggested a Th2 shift [23,62]. Here,
we found a substantial downregulation of the Th1 response in
BTBR mice, as compared to B6 mice; IL-12 and IFNc expression
was similar or lower, whereas IL-33 and IL-10, cytokines
promoting Th2-mediated response [22,64], were elevated. The
greater IL-6 expression in the brain of BTBR mice may be worth
noting since IL-6 is involved in sickness behavior and also in
modulation of brain development (probably mediated by neuronal
inflammation) [69,70]. In addition, IL-6 has been reported to
mediate the behavioral abnormalities of adult offspring from dams
that experienced maternal immune activation during pregnancy
. Since type-2 immunity appears to predominate in BTBR
mice and IL-33, a type-2 immune promoter, is the only cytokine
significantly elevated in all brain regions, IL-33 may be especially
important to consider for its affects on behavior, in that it has been
suggested that IL-33-producing glia are critical regulators of innate
immune responses in the CNS .
The existence of an animal model will be very valuable in the
investigation of mechanistic pathways and therapeutic interven-
tions of psychiatric diseases, especially for neurodegenerative or
neurodevelopmental disorders. The present study is the first report
of the involvement of neuroinflammation, accompanied by
elevation of IgG and IgE and IgG-secreting B cells, in BTBR
mice, a strain with abnormal behaviors. Considering the elevated
amount of IgG accumulated in the brain and serum anti-brain
antibodies of BTBR mice, specific brain Ag(s) need to be
identified, and the involvement of humoral immunity in the
aberrant behaviors of the BTBR mice needs to be explored. The
Table 6. Levels of IgE in brain regions.a
Level of IgE (ng/mg protein)b
StrainSex Cortex (CTX)c
B6Male1.860.21.760.35.861.1 1.860.211.961.542.564.8 2.860.2
Female1.560.21.460.211.360.6 1.260.2 12.561.633.4126.96.36.199
aEach brain region was isolated from perfused postnatal day 21 mice, and the homogenates were used for evaluation of IgE level present in each region. Number of
mice was 4 for each sex and strain. The results are expressed as mean 6 SEM.
bIgE levels in brain regions of the mouse strains were significantly different by three-way ANOVA with BTBR and B6 mice with SN and HT being the most significantly
cTwo-way ANOVA indicates a strain difference; Bonferroni t-test indicates that BTBR differs from B6 (p,0.001).
dTwo-way ANOVA indicates a sex difference; Bonferroni t-test indicates that males differ from females (p,0.001).
eTwo-way ANOVA indicates strain and sex differences; Bonferroni t-test indicates that male and females differ within strain and strains differ (p,0.001).
Figure 11. Correlation analysis of brain IgG and IL-33 of the
strains with their behavior index.
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genetic basis for the constitutively high levels of IgG and IgE also
need to be investigated. Although higher levels of several
proinflammatory cytokines, including IL-33, IL-18, and IL-1b,
were shown in BTBR in the present study, brain cellular sources of
these cytokines need to be delineated. It also is necessary to
evaluate the individual contribution of each cytokine to the
development of the aberrant BTBR behaviors, as well as the cross-
talk between proinflammatory cytokines and certain anti-inflam-
matory cytokines, such as TGFb1. Since BCF1 mice (born of
BTBR dams, sired by B6), especially the males, demonstrated
immunologic characteristics closer to BTBR mice than did the
CBF1 mice, these combinations may be useful for dissection of the
genetic and environmental influences on development of the
Materials and Methods
BTBR breeder mice were kindly provided by Dr. Valerie J.
Bolivar (Wadsworth Center, New York State Department of
Health). BTBR and B6 mice also were purchased from Jackson
Laboratories (Bar Harbor, ME), and BALB/cByJ and BALB/c-
scid mice (mice lack B cells and T cells and thus have no IgG) were
obtained from the Wadsworth Center’s Animal Production Unit.
BTBR and B6 mice are both H-2b. Mice were housed in sterile
laminar flow cages in a specified pathogen free facility of the
Wadsworth Center and were maintained on autoclaved food and
water. All of our animal maintenance, breeding, and experimental
procedures were approved by the Wadsworth Center’s IACUC,
Protocol # 09-278. F1mice were derived from BTBR females6B6
males (BCF1) or B6 females6BTBR males (CBF1). All of the mice
used were sacrificed at pnd21 or after behavioral analysis at
pnd70. All of the experimental data were obtained from at least
three separate litters for each strain and both genders.
Preparation of brain tissue homogenates for IgG or
Whole brain or various brain regions (cortex, striatum,
hippocampus, hypothalamus, substantia nigra, and cerebellum)
were collected from pnd21 mice following intracardial perfusion
with warm phosphate-buffered saline (PBS; 50 ml/mouse). Each
brain was dissected with microdissection forceps and scissors. The
brain tissues were homogenized in the extraction buffer containing
20 mM Tris, 100 mM NaCl, 2 mM Na2EDTA, 1% NP-40, and
protease inhibitor cocktail (Sigma, St. Louis, MO), followed by
sonication for 10 min. The supernatants were collected following
30 min centrifugation (16,000 g) at 4uC, and kept frozen before
use. The homogenate protein concentration was determined with
BCA protein assay kit (Pierce, Rockford, IL).
The level of total IgG in serum or brain homogenates was
determined by a sandwich ELISA using goat anti-mouse IgG Fc
(Pierce, Rockford, IL) as a capture Ab and HRP-goat anti-mouse
IgG as a detection Ab (Sigma). Brain homogenates and sera were
diluted 1/100 and 1/100,000, respectively, with PBS.
Presence of brain-reactive IgG in mice sera was determined as
follows . SCID mouse whole brain or brain regional proteins
were coated (10 mg/well) in the 96-well plate overnight at 4uC,
and after 36washing, the plate was blocked with 5% BSA-PBS for
2 h. SCID brains were used so as to ensure the absence of IgG in
the brain homogenates. Sera from pnd21 mice were added into
the wells, and incubated overnight at 4uC. After 66 washing,
HRP-goat anti-mouse IgG detection Ab was added and incubated
for 2 h at room temperature. The plates were washed 66, and the
BD PharmingenTMTMB substrate solution (San Diego, CA) was
added for color development. Absorbance was measured at
450 nm, with 570 nm as the reference wavelength. SCID serum
was used as a negative control.
ELISPOT analysis of immunoglobulin-secreting cells
MultiScreen Filtration plate (Millipore, Billerica, MA) was pre-
wetted with 50 mL of 70% ethanol in sterile PBS per well. After 26
wash with sterile PBS, the plate was coated with goat anti-mouse
IgG c chain (Mabtech, Cincinnati, OH) as capture Ab (2 mg/
100 mL/ well) in sterile PBS at 4uC overnight. The plate was
washed 56with PBS, and then RPMI 1640 medium containing
5% BSA was used to block the plate at 37uC for 2 hr. The
blocking medium was discarded, and 105splenic cells in 100 mL of
complete RPMI 1640 medium were added to each well and
incubated at 37uC with 7% CO2, 5% O2, 88% N2, and high
humidity for 24 hr. The cells were then discarded, and the plate
was washed 66 followed by addition of biotinylated goat anti-
mouse IgG (0.2 mg/100 mL/well in PBS containing 0.05% tween
20 and 1% BSA; Mabtech) and streptavidin-ALP ( 100 mL/ well;
1:1000 diluted with PBS; Mabtech). After additional washings, the
plate was incubated with the substrate BCIP/NBT for 15 min at
room temperature. Then the plate was washed extensively with
water and dried in the dark. The next day the plate was read with
an ImmuoSpot analyzer (Cellular Technology, Shaker Heights,
Nine cytokines (IL-33, IL-18, IL-1b, IL-6, IL-10, IFNc, IL-12,
IL-2, TNFa) were assayed for expression in brain homogenates
from pnd21 pups. IL-33 was quantified with the DuoSet ELISA
set (R&D, Minneapolis, MN). IL-18 was assayed using rat anti-
mouse IL-18 Ab and biotinylated rat anti-mouse IL-18 Ab, as a
paired capture and detection Ab (R&D). The levels of the other
cytokines were evaluated with a Fluorokine MAP mouse multi-
analyte profiling kit (R&D) and analysis with a Luminex 100
(Austin, TX). The respective lower limits of detection were 1.21,
0.2, 0.078, 4.0, 1.81, 0.65, and 0.01 pg/ml, for IL-1b, IL-6, IL-10,
IFNc, IL-12, IL-2, and TNFa, respectively.
Flow cytometric analysis
Single cell suspensions were prepared from spleens or
mesenteric lymph nodes as described elsewhere [73,74]. Three
mesenteric lymph nodes were collected from each mouse.
Mononuclear cell populations in the spleens or lymph nodes were
determined by four color fluorescence-activated cell sorting
(FACS) with a FACSCalibur flow cytometer (BD Biosciences).
Anti-CD45-PerCP, anti-CD3-APC, and anti-CD4-FITC Abs
were used for helper T cell phenotyping, and instead of anti-
CD4-FITC, anti-CD8-FITC or anti-CD19-FITC, anti-CD5-APC
Ab was used for cytotoxic T cell or B cell phenotyping,
respectively. .Anti-CD40-PE and anti-I-A-PE Abs also were used
for B cells. In addition to anti-CD45-PerCP and anti-CD3-APC
Abs, anti-CD19-FITC and anti-CD138-PE Abs were added for
enumeration of a plasma cell population. Absolute numbers and
percentages of these cell populations in whole blood were
determined with use of BD TruCOUNTTMtubes (50 ml blood/
Preparations of brain single-cell populations containing microg-
lial cells, dendritic cells, and mast cells were performed as
described [75,76], with a modification. In brief, after perfusion
with PBS, brains were removed, incubated with liberase (0.2 mg/
brain, Roche, Indianapolis, IN) for digestion, and minced; use of
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liberase for obtaining single cell suspensions of brain cells was
previously described . The homogenized brain samples were
passed through 55 mm diameter nylon mesh. The resulting
suspensions were centrifuged at 250 g for 8 min, and the pellet
was resuspended in 40% Percoll (GE Healthcare, Piscataway, NJ)
solution, followed by centrifugation at 800 g for 30 min. The
resulting pellet was washed with 40 ml of 3% FBS-PBS through
centrifugation, and then resuspended in PBS with 0.1% sodium
azide. Four-color FACS was applied to determine the proportions
of the various brain cell subpopulations by a FACSCanto (BD
Biosciences) flow cytometer. Anti-CD45-APC-Cy7, anti-CD11b-
PE-Cy7 (BD Biosciences), and anti-F4/80-Alexa Fluor 488
(Serotec, Oxford, UK) Abs were used for identification of
microglial cells. Instead of anti-F4/80-Alexa Fluor 488 Ab, anti-
CD11c-FITC or anti-FceR1-FITC (eBioscience, San Diego, CA)
Ab was used for enumeration of brain dendritic cells or mast cells,
respectively. TO-PRO-3 (Invitrogen, Carlsbad, CA) was used to
define viable cells.
For the evaluation of IgG deposition in the brain, we utilized
perfused brains; 200 mm sections were washed in wells of a 24-well
culture plate with 6 washes with PBS to remove IgG not specific to
a brain antigen. The sections were then fixed 4% paraformalde-
hyde in PBS for 1 hr at room temperature (RT) followed by 3
washes with PBS. The sections were then blocked with 3% goat
serum in PBS for 30 min at RT and stained with 488-conjugated
chicken anti-mouse IgG (1:300 diluted with 3% goat serum in
PBS) overnight at 4uC. The sections were washed 6 times, placed
on charged slides and assayed by fluorescence microscope.
Toluidine Blue staining and Digital Image Acquisition
Mice were perfused with PBS followed by 4% PFA, then
sacrificed by CO2 asphyxiation. Brains were immediately
removed, rinsed in saline and then immersion-fixed in 4%
paraformaldehyde for 48 hr at 4uC. Fixed brain tissues were
paraffinized using a Tissue Tek processor, and sectioned at 6 mm
using a rotary microtome. In this study, tissue sections at the level
of the forebrain caudate nucleus (Bregma +1.4 mm), rostral
hippocampus (Bregma 21.3 mm), medial hippocampus (Bregma
22.5 mm) and cerebellum (Bregma 26.12 mm) were stained with
toluidine blue to ascertain whether there were region and strain
differences in mast cell density and morphology between BTBR
and B6 mice. Briefly, for mast cell identification, tissue sections
were cleared in xylenes, prior to rehydration through graded
alcohols to distilled water. Toluidine blue staining solution was
freshly prepared, and comprised 5 ml of toluidine stock solution
(1 g toludine blue – Sigma in 100 ml of 70% alcohol) with 45 ml
of 1% Sodium Chloride acidified solution at pH 2.2. Sections were
immersed in toluidine blue for 10 sec then rinsed 3 times in
distilled water. Sections were then dehydrated through graded
alcohols, cleared in xylenes prior to mounting with DPX (sigma)
and coverslipping. For digital image acquisition, regions of interest
were identified through comparison with a sterotaxic mouse brain
atlas of Paxinos and Franklin . Digital photo-micrographs
were generated using a Nikon (50i) microscope using 106 and
606lens coupled to a CCD camera with image grabber software.
Mast cells were clearly visible as metachromatic purple color on an
orthochromatic blue background.
Blood Brain Barrier (BBB) permeability test
The BBB test for pnd21 mice was performed through
examination of vascular leakage of Evans blue (EB) dye . EB
dye solution (50 mg/g body weight) was intraperitoneally injected
into mice, and after 5 h, the mice were anesthetized with CO2gas,
and cardiac blood was collected for plasma separation. Brains
were removed after PBS perfusion and weighed. Formamide
(0.5 ml/brain, Sigma-Aldrich) was added to the tube containing
brain, for extraction of EB dye from brain. The tubes were
centrifuged at 16,000 g for 15 min for collection of supernatants
after 72 h incubation with formamide. Optical density (OD) of the
supernatants or plasma was measured at 620 nm by an
absorbance microplate reader (Bio-Tek, Winooski, VT). The
OD of pure formamide solution was subtracted as a background
absorbance from each sample’s OD (sample’s adjusted OD). The
adjusted ODs of brain samples were again adjusted to reflect
weight differences among brain samples (adjusted OD). Finally,
the BBB permeability index was calculated via division of the
adjusted OD of each brain sample by the adjusted OD of the
matching plasma sample followed by multiplication by 10.
Critical to the validity of any biochemical, immunological, or
molecular analyses for the autism-like assessments is the behavioral
test [15,80]. The sociability task measures the tendency of the
subject mouse to approach the stimulus (novel) mouse, as well as
the time spent with the mouse; these sessions include specific
behaviors such as sniffing and licking. The novel mouse (BALB/
cByJ) used is genetically different from the test mice, but the same
gender, and is used only for one test per day. Behaviors of test mice
were assayed at 8–10 wk of age. The stimulus mouse is enclosed
inside a small circular wire cage. Thus, the stimulus mouse can
provide visual, olfactory, auditory and some physical contact cues.
An identical empty wire cage is placed in the other adjacent
chamber. At the end of the habituation period, the dividers are
removed so that the subject mouse can explore either of the
adjacent chambers. The testing session is 10 min long. If the
subject mouse is ‘‘sociable’’ it will spend more time in the chamber
with the stimulus mouse. If not, it will either spend more time in
the other adjacent chamber or remain in its central chamber. The
calculated ‘‘behavior index’’ is the time spent with the mouse
minus the time spent with the object.
Mice were immunized with KLH (100 mg in 200 ml saline). One
week later, blood was collected by retro-orbital phlebotomy for
sera and the mice were boosted with 100 mg KLH in 200 ml saline.
Blood was again collected after 1 week. The sera were assayed for
IgM anti-KLH, IgG1 anti-KLH, IgG2a anti-KLH, IgE anti-KLH,
and total IgE by a standard ELISA assay, as described previously
. Briefly, KLH (10 mg/ml) in 0.1 M carbonate pH 9.5 (8.4 g
NaHCO3, 3.56 g Na2CO3, in 1 L) was coated onto ELISA plates
overnight at 4uC. After blocking with PBS plus 1% BSA, a serial
dilution of the internal control (mouse IgG2a anti-KLH; Sigma) or
serum was added. Biotinylated detection mAbs (biotin anti-mouse
IgG1, IgG2a, or IgM from BD Science) were applied followed by
Avidin-peroxidase (PharMingen) and substrate. The plates were
read using an ELISA reader (EL310, Bio-Tek, Burlington, VT) at
450 nm. For measurement of KLH-specific IgE, 2 mg/ml purified
anti-mouse IgE (PharMingen) was coated overnight at 40C. After
blocking with PBS plus 1% BSA, a serial dilution of the internal
control (mouse IgE, PharMingen) and serum was added. Then,
KLH (10 mg/ml) was added into sample wells only. After 2-hr
incubation at room temperature, mouse IgG2a anti-KLH,
followed by Biotin anti-mouse IgG2a, avidin-peroxidase was
applied. For the internal control development, 2 mg/ml biotin
anti-mouse IgE (PharMingen) and avidin-peroxidase were used.
The same method was utilized for color development and for
Immunity of BTBR T+tf/J Mice
PLoS ONE | www.plosone.org13 July 2011 | Volume 6 | Issue 7 | e20912
reading of the plates. For total IgE, the OptEIA Mouse IgE Set
(PharMingen) was employed. The protocol provided by the
manufacturer was used.
Host resistance against Listeria monocytogenes
Host resistance against Listeria monocytogenes (LM) was measured
as previously described . Briefly, adult mice were injected
intravenously with 105viable LM. Mice were weighed daily for
assessment of sickness behavior, measured as lack of eating and
drinking leading to weight loss. At day three after infection (the
peak response), livers and spleens were aseptically harvested for
assessment of bacterial burden by enumeration of LM colony
forming units (CFU); serial dilutions of organ homogenates are
plated in quadruplicate on blood-agar plates. Bacterial burdens
are expressed as CFU per organ.
The data were expressed as mean values 6 SEM, and initially
evaluated by three-way, two-way, or one-way ANOVA (gender,
strain); however, since there were no significant gender differences,
some of the data were further evaluated for normal distributions
by one-way ANOVA and post-hoc tests. Differences with p,0.05
were considered significant. Analyses utilized SigmaPlot 11.
We thank Dr. Valerie Bolivar for her generous donation of BTBR mice, for
introducing us to the BTBR mouse model, and for training in the
Behavorial Core of Wadsworth Center. We also thank the Immunology
Core facility (Joan Pedersen-Lane and Renji Song), the Clinical Cellular
Immunology Unit (Dr. Maria Lopez, Dr. Jane Kasten-Jolly, and Nancy
Andersen) of the Wadsworth Center for assistance with flow cytometry and
Luminex analysis. We thank Adriana Verschoor for her editorial
Conceived and designed the experiments: YH YZ DG DAL. Performed
the experiments: YH YZ DG VMM. Analyzed the data: YH YZ DG
VMM DAL. Wrote the paper: YH YZ VMM DAL.
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