Anti-Gluten Immune Response following Toxoplasma
gondii Infection in Mice
Emily G. Severance1*, Geetha Kannan2, Kristin L. Gressitt1, Jianchun Xiao1, Armin Alaedini3,
Mikhail V. Pletnikov2, Robert H. Yolken1
1Stanley Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of
America, 2Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America,
3Department of Medicine, Columbia University Medical Center, New York, New York, United States of America
Gluten sensitivity may affect disease pathogenesis in a subset of individuals who have schizophrenia, bipolar disorder or
autism. Exposure to Toxoplasma gondii is a known risk factor for the development of schizophrenia, presumably through a
direct pathological effect of the parasite on brain and behavior. A co-association of antibodies to wheat gluten and to T.
gondii in individuals with schizophrenia was recently uncovered, suggesting a coordinated gastrointestinal means by which
T. gondii and dietary gluten might generate an immune response. Here, we evaluated the connection between these
infectious- and food-based antigens in mouse models. BALB/c mice receiving a standard wheat-based rodent chow were
infected with T. gondii via intraperitoneal, peroral and prenatal exposure methods. Significant increases in the levels of anti-
gluten IgG were documented in all infected mice and in offspring from chronically infected dams compared to uninfected
controls (repetitive measures ANOVAs, two-tailed t-tests, all p#0.00001). Activation of the complement system
accompanied this immune response (p#0.002–0.00001). Perorally-infected females showed higher levels of anti-gluten
IgG than males (p#0.009) indicating that T. gondii-generated gastrointestinal infection led to a significant anti-gluten
immune response in a sex-dependent manner. These findings support a gastrointestinal basis by which two risk factors for
schizophrenia, T. gondii infection and sensitivity to dietary gluten, might be connected to produce the immune activation
that is becoming an increasingly recognized pathology of psychiatric disorders.
Citation: Severance EG, Kannan G, Gressitt KL, Xiao J, Alaedini A, et al. (2012) Anti-Gluten Immune Response following Toxoplasma gondii Infection in Mice. PLoS
ONE 7(11): e50991. doi:10.1371/journal.pone.0050991
Editor: Ira Blader, University of Oklahoma Health Sciences Center, United States of America
Received May 24, 2012; Accepted October 29, 2012; Published November 29, 2012
Copyright: ? 2012 Severance 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 a National Institute of Mental Health (NIMH) P50 Silvio O. Conte Center at Johns Hopkins (grant# MH-94268; http://www.
nimh.nih.gov/); by the Brain and Behavior Research Foundation where Dr. Severance is a Scott-Gentle Foundation Young Investigator (http://bbrfoundation.org/);
and by the Stanley Medical Research Institute (www.stanleyresearch.org/). These funders played 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: firstname.lastname@example.org
Gluten proteins of wheat and related cereals have a pathogenic
effect on the intestinal tract of individuals with the autoimmune
disorder, celiac disease . Increasingly, the exposure to gluten
and to other food antigens such as bovine milk caseins is
implicated in the pathogenesis of neuropsychiatric diseases,
[2,3,4,5,6,7,8,9,10,11,12,13,14]. The digestion of wheat glutens
and milk caseins has been proposed to result in the production of
neuroactive exorphins that penetrate compromised gastrointesti-
nal (GI) and blood brain barriers and directly bind to opioid
receptors in the CNS [4,8,10,15,16,17,18]. We recently reported
significant correlations of markers of GI inflammation with
antibody levels to gluten and casein in individuals with schizo-
phrenia . Among these correlations, we found that antibodies
to the targeted food antigens were associated with antibodies to the
protozoan parasite, Toxoplasma gondii, in individuals with a recent
onset of the disease.
T. gondii has been studied predominantly in the context of
psychiatric disorders as a pathogen that might modify host
behavior through a direct effect on the central nervous system
[19,20,21,22,23,24,25,26,27,28]. The association between food
antigen exposure and the T. gondii parasite in individuals with
schizophrenia presents a peripheral pathway via the GI tract by
which this parasite may also contribute to psychiatric disease. In
murine models, the oral ingestion of T. gondii can cause small
intestinal immunopathology and is thus used experimentally to
induce colitis and ileitis [29,30,31,32,33,34]. In humans, increased
titers of T. gondii antibodies have been found in individuals with
inflammatory bowel disease and celiac disease . In pregnant
women, T. gondii infection rates were higher among those with
celiac disease compared to those without a gluten sensitivity .
It is not possible to discern from these studies if GI pathologies
preceded T. gondii infection or vice versa.
Here, we investigated the functional link between two previously
unassociated schizophrenia risk factors, T. gondii exposure and
immune reaction to gluten, in experimental mouse models. We
also evaluated if adult and prenatal exposures to T. gondii might
impact complement activation, a marker of systemic inflammation
and antigen presence in the circulation [37,38,39,40]. Results
from these experiments demonstrate for the first time that
intestinal T. gondii infection can lead to elevated antibody levels
to dietary gluten. These findings lay the groundwork for future
PLOS ONE | www.plosone.org1 November 2012 | Volume 7 | Issue 11 | e50991
studies to explore how digested gluten peptides, independently or
combined with T. gondii and other GI pathogens, might impact
brain physiology and behavior.
Materials and Methods
Investigations were approved by the Johns Hopkins Animal
Care and Use Committee Institutional Guidelines (permit
#MO12M23). These studies were part of a larger ongoing project
to evaluate genetic and environmental hypotheses of schizophre-
nia in mouse models. Utmost effort was utilized to prevent
suffering and minimize the numbers of mice required for each
Exposure of mice to T. gondii was achieved using three routes of
infection: intraperitoneal (IP), peroral (PO), and prenatal. An
overview of how the experimental groups were generated via each
type of T. gondii infection is depicted in Figure 1.
Twenty-four 8-week old Balb/C mice were injected into the
peritoneal cavity with either 400 tachyzoites of the Type II
T. gondii strain, Prugniaud (PRU; n=6 male; n=6 female), or with
Dulbecco’s phosphate buffered saline (DPBS; mock-infected; n=6
male; n=6 female). Serum samples were collected from a tail clip
at 0, 15 and 21 days post-infection (dpi). IgG levels to T. gondii,
gluten and complement factor C1q were measured with enzyme-
linked immunosorbent assays (ELISAs), as described below. Over
the course of the study, four of the female IP T. gondii-infected mice
died. These animals were not included in the analyses.
A PO-infected cohort was generated with the goal of providing
a more natural means of T. gondii infection and limiting to the
extent possible any stress introduced by the procedure itself. Thus,
in this PO model, we developed a diet containing infectious
T. gondii mixed with a crushed rodent chow that contains as its
primary protein ingredient ground wheat (2018SX Teklad Global
18% Protein Extruded Rodent Diet; Harlan Laboratories,
Madison, WI, U.S.A.). Animals were then allowed to eat freely
(i.e. they were not forced). This PO method of inoculation was
expected to produce infection in only a subset of animals that
received this diet.
To prepare infectious T. gondii material, 5- and 8-week old
BALB/c and mixed background BALB/c crossed with C57BL6,
F4 donor mice were injected IP with 400 tachyzoites of the
T. gondii PRU strain. At five weeks post-infection, mice were
sacrificed, their brains harvested, and serum obtained from a tail
clip. Infectious brain homogenates were then mixed with crushed
Recipient mice were composed of two cohorts of BALB/c mice.
Cohort 1 included twelve 12-week old mice (n=6 male; n=6
female); cohort 2 included ten 4-week old (n=5 male; n=5
female) and ten 6-week old mice (n=5 male; n=5 female).
Recipient mice were food-deprived for 19 hours prior to infection.
The food mixture of brain homogenate and crushed rodent chow
was placed in the cages of all recipient mice, and mice were
allowed to freely eat for three hours. Serum was collected at 4–5
weeks post-infection for cohort 1 and at 3 weeks post-infection for
the cohort 2. These two cohorts were combined for the data
analyses described below. Antibodies to T. gondii, gluten and C1q
were measured via the ELISAs described below.
Female mice were injected IP with 400 tachyzoites of the
T. gondii PRU strain. T. gondii-seropositive dams were mated with
uninfected males. Eleven offspring were produced from this
mating. The control group was composed of DPBS mock-infected
dams mated with uninfected males. Four offspring resulted from
this mating. Serum was taken from all offspring from a tail clipping
at postnatal day 7. Antibodies to T. gondii, gluten and C1q were
measured as described below.
Laboratory ELISA Procedures
In serum samples, IgG antibodies to T. gondii, gluten and C1q
were measured by ELISAs. Commercially available ELISA kits for
measuring serological T. gondii IgG were purchased from IBL
America (Minneapolis, MN, U.S.A.) and used as previously
described with a 1:50 serum dilution and an anti-mouse IgG
secondary antibody . IgG antibodies to wheat gluten and C1q
were measured by ELISAs using previously described methods
with some modification [11,42]. Whole gluten was extracted from
the wheat cultivar Cheyenne, also as previously described .
C1q was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.)
Wells of 96-well microtiter plates were incubated with one
hundred ng of protein in 50 ml carbonate buffer overnight at
4uC. The plates were incubated with serum samples diluted 1:50
in PBST for 2 h at 37uC. Plates were washed and incubated with
peroxidase-conjugated goat-anti-mouse IgG secondary antibody
for 30 min at 37uC (Southern Biotech, Birmingham, AL, U.S.A.).
peroxide solution (KPL Protein Research Products, Gaithersburg,
MD, U.S.A.) was added for color development, and absorbance
was measured at 405 nm, with a reference wavelength of 490 nm,
in an automated microtiter plate reader (Molecular Devices,
Menlo Park, CA, U.S.A.).
Using the above ELISA methods, standard curves of antibody
levels (mg/ml) were generated for T. gondii using the calibrators
available in the commercial kit and for gluten using a rabbit anti-
gliadin polyclonal antibody (Sigma-Aldrich, St. Louis, MO,
U.S.A.). The concentration of antibodies specific to T. gondii or
to gluten was then calculated. For the T. gondii calibrators provided
in the commercial kit, conversion of International Units to
milligrams per milliliter was performed according to Humphrey
and Batty . Standard curves for determination of serum IgG
estimates are found in Figure 2.
T. gondii seropositivity was established based on values from the
ELISA kit control standards run with each assay. Anti-T. gondii, -
gluten and -C1q IgG levels were compared between: (1) IP
T. gondii-infected and IP mock-infected animals, (2) PO-infected
T. gondii-seropositive and PO-infected T. gondii-seronegative ani-
mals, and (3) offspring of infected vs offspring of uninfected
mothers. Repetitive measures ANOVAs were applied in the IP
experiment to identify significant differences in antibody levels
over time among groups according to sex and infection status.
Statistical differences in mean levels of antibodies among groups
and sex-specific subgroups in the PO and prenatal exposure
cohorts were identified with two-tailed t-tests. P-values less than
0.05 were considered to be statistically significant. Statistical
analyses were performed with STATA version 12 (STATA Corp
LP, College Station, Texas, U.S.A.).
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org2 November 2012 | Volume 7 | Issue 11 | e50991
Figure 1. Overview of modes of T. gondii infection and comparison groups. Panel A: Adult mice received intraperitoneal injections as shown
to generate a T. gondii-infected and a mock-infected group. Experimental groups are boxed in red. Panel B: Adult mice received diets containing T.
gondii, which resulted in mice that were successfully infected with T. gondii (T. gondii-seropositive) and those that were exposed to but did not
develop an infection (T. gondii-seronegative). Panel C: IP- and mock-infected females were mated to uninfected males to produce offspring that were
(1) T. gondii-exposed and T. gondii-seropositive, (2) T. gondii-exposed and T. gondii-seronegative, or (3) uninfected.
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org3 November 2012 | Volume 7 | Issue 11 | e50991
Intraperitoneal (IP) Infection
We first measured anti-T. gondii IgG levels to establish T. gondii-
seropositive and T. gondii-seronegative groups. Following IP
infection, highly statistically distinct T. gondii-seropositive and
T. gondii-seronegative groups were generated. The IP route of
infection resulted in T. gondii seropositivity in 100% of the animals
that were inoculated with the parasite (n=6 males; n=6 females).
Animals injected with a DPBS vehicle (mock-infected; n=6 males;
n=6 females) were T. gondii-seronegative. Four of the six females
from the IP T. gondii group died during the course of the
experiment, and these animals were not included in the analyses.
Anti-T. gondii IgG levels increased over time in T. gondii-inoculated
animals compared to those that were mock-infected (Figure 3,
p#0.00001; interaction of infection group and time since
infection, p#0.00001). There were no differences in T. gondii
IgG between sexes in either the T. gondii-infected or mock-infected
In IP-inoculated mice, anti-gluten IgG increased over time in
animals infected with T. gondii (n=8) compared to those that were
mock-infected (n=12; Figure 3, panel B; repetitive measures
ANOVA, between groups p#0.003; interaction of infection group
and time since infection, p#0.00001). When animal groups were
broken down according to sex, the interaction of infection group
Figure 2. Standard curves from ELISAs for estimation of serological levels of anti-T. gondii and anti-gluten IgG. Panel A: The amount of
T. gondii calibrator antibody is plotted according to absorbance. The standard curve shown is defined by the equation, y=0.5342ln(x)+1.0478. The
range of detection of T. gondii antibody for this assay is 100 ng-20 mg/ml. IP and PO inoculations produced T. gondii antibodies in excess of 20 mg/
ml. Prenatal exposure to T. gondii resulted in IgG levels of around 1 mg/ml in offspring. Panel B: The amount of gluten antibody is plotted according
to absorbance. The gluten antibody curve is defined by the equation, y=0.235ln(x)+0.8572. The range of detection for this assay is 20 ng–2 ug/ml. IP,
PO and prenatal exposures resulted in IgG levels of 50–90 ng/ml serological anti-gluten IgG. T1, T2, T3 refer to replicates of the assays run to
construct these standard curves.
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org4November 2012 | Volume 7 | Issue 11 | e50991
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org5 November 2012 | Volume 7 | Issue 11 | e50991
and time remained significant for the males (n=12; between
infection groups, p#0.0002; interaction p#0.00001), but not
females (n=8; infection groups, p#0.49; interaction p#0.20).
This difference between sexes in gluten antibody levels over time
was attributable to the mock-infected group (Figure 3, panel B; sex
p#0.008; interaction sex and time, p#0.04), whereas no
difference in anti-gluten IgG levels over time was observed
between sexes in the T. gondii-infected animals (Figure 3, panel B;
sex p#0.69; interaction sex and time, p#0.72).
Complement C1q activation in IP-inoculated mice was
significantly elevated compared to mock-infected mice and this
response increased over time since infection (Figure 3, panel C;
between infection groups p#0.00001; interaction p#0.00001;
n=7 T. gondii-infected, n=11 mock-infected). Levels of C1q
activation were not significantly different between sexes in the
T. gondii- or mock-infected groups.
Peroral (PO) Exposure
In animals exposed to T. gondii in their diet (PO), ten of 12 mice
from the first cohort (12 week-old mice tested 4–5 wpi) and eight of
20 mice from the second cohort (4- and 6-week old mice tested 3
wpi) tested seropositive for T. gondii. The PO rate of infection using
this method was thus 56.25%. Seropositive mice had significantly
higher levels of anti-T. gondii IgG than those that were seronegative
(Figure 4, panel A; two-tailed t-test, t=21.7e02, p#0.00001).
There were no significant differences in anti-T. gondii IgG between
sexes (seropositive, n=9 male, n=9 female; seronegative, n=7
male, n=7 female).
In animals orally exposed to T. gondii, mean levels of anti-gluten
IgG were significantly greater in T. gondii-seropositive (n=18) vs
T. gondii-seronegative (n=14) mice (Figure 4, panel B; two-tailed t-
test, t=25.16, p#0.00001). In the T. gondii-seropositive group,
anti-gluten IgG levels were significantly elevated in females
compared to males (Figure 4, panel B; two-tailed t-test
t=23.00, p#0.009). Gluten antibody levels were not significantly
different between sexes in the mock-infected group (Figure 4,
panel B; two-tailed t-test, t=0.74, p#0.48).
C1q antibody levels were significantly increased in T. gondii-
seropositive mice (n=14) compared to those that were seroneg-
ative (n=8; Figure 4, panel C; two-tailed t-test t=26.44,
p#0.00001). No sex-specific differences were detected.
Sexes were not determined for the 7-day old pups born in the
prenatal cohort. Of the 11 mice born to the infected mothers,
100% were T. gondii-seropositive. The four mice born to
uninfected mothers were T. gondii-seronegative. T. gondii IgG
levels were significantly increased in the seropositive group
(Figure 5, panel A; two-tailed t-test t=28.91, p#0.00001).
Mean gluten IgG levels of pups (n=11) born to T. gondii-
seropositive dams were significantly greater than levels of pups
(n=4) born to uninfected dams (Figure 5, panel B; two-tailed t-
test, t=27.73, p#0.00001).
C1q antibodies were significantly increased in offspring of
T. gondii-seropositive dams compared to offspring from seronega-
tive dams (Figure 5, panel C; two-tailed t-test, t=23.7, p#0.002).
Our findings convincingly indicate a direct relationship between
infection with T. gondii and the generation of antibodies to gluten.
A standard laboratory rodent chow contained enough wheat to
precipitate the anti-gluten immune response in infected animals.
In particular, female mice infected perorally with T. gondii had a
significantly stronger anti-gluten immune response compared to
infected males or uninfected control animals. Female-specific
effects were also found in IP-inoculated animals; however, this
method of infection was associated with a high mortality rate and
is itself a less natural means of infection by T. gondii than the
peroral route. The anti-gluten immune response and activation of
the complement system following prenatal exposure to T. gondii
infection coincides with a critical time period of postnatal neuronal
development. These results, therefore, offer important milestones
for future experiments to evaluate how the combined exposure to
infection and dietary antigens, both known risk factors for the
development of schizophrenia, might contribute to neurodevelop-
mental hypotheses of psychiatric disease etiology.
Results from both the PO and IP portions of our study provide
support that the female sex is more severely affected following T.
gondii infection. Four out of the six females inoculated IP with T.
gondii died, compared to100% viability in males infected using the
same method. Indeed, others have shown that female mice are
more susceptible to and more likely to die from T. gondii infection
than males [44,45]. Therefore, perhaps it is not surprising that PO
T. gondii launched a stronger anti-gluten immune activation in
female mice compared to similarly infected males. Even in the IP
mock vehicle group of our study, females exhibited significant
elevations of gluten antibodies compared to pre-injection levels;
males in this group showed no significant anti-gluten immune
activation. This sex bias following a seemingly innocuous
laboratory procedure is in keeping with reports from several
groups regarding differential immune responses by female vs. male
BALB/c mice [46,47,48]. Drude and colleagues (2011) reported
that BALB/c mice undergoing standard laboratory injections with
non-toxic substances such as saline and cyclodextrin exhibited sex-
specific differences in the stress response, which in turn resulted in
stress-induced lymphocytopenia . Our results confirm that
routine laboratory methods such as the IP route of delivery are not
benign, and that stress-induced immune activation might, in fact,
be compounded by exposure to dietary food antigens. Gluten
antibody elevations in the IP mock-infected group of female mice
further illustrate that it is the integrity of the GI environment that
contributes to this food antigen response, a condition that can be
brought on by stress or as modeled here by T. gondii infection. It is
likely that infection with other GI pathogens would also cause a
level of GI dysfunction that would directly and/or indirectly
through associated immune activation/inflammation, affect the
intake of nutrients and/or production of dietary antigens. As such,
Figure 3. IgG antibody levels following intraperitoneal (IP) T. gondii inoculation. Panel A: Levels of anti-T. gondii IgG in mice infected IP
with T. gondii (red) varied significantly over time compared to mock-infected control mice (blue). T. gondii antibody levels were not different between
sexes (hashed bar – males; open bar – females). Panel B: Levels of anti-gluten IgG in mice infected IP with T. gondii varied significantly over time
compared to mock-infected control mice. Between mock-infected male and female mice, anti-gluten IgG levels varied significantly over time. In IP T.
gondii-infected mice, anti-gluten IgG levels over time were not significantly (ns) different between sexes. Panel C: Levels of anti-C1q IgG in IP-infected
mice varied significantly over time compared to mock-infected control mice. Time, infection and sex refer to interaction variables in repetitive
measures ANOVAs. Repetitive measures ANOVAs were used to generate listed p-values. Error bars indicate standard errors of the mean. NS refers to
not significant. Dpi refers to days post-infection.
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org6 November 2012 | Volume 7 | Issue 11 | e50991
Figure 4. IgG antibody levels following peroral (PO) T. gondii exposure. Panel A: Levels of anti-T. gondii IgG were significantly greater in mice
that were seropositive for T. gondii (T. gondii-positive) compared to those that were seronegative (T. gondii-negative) following ingestion of rodent
chow containing T. gondii. Panel B: Levels of anti-gluten IgG were significantly greater in mice that were seropositive for T. gondii (T. gondii-positive)
compared to those that were seronegative (T. gondii-negative) following ingestion of rodent chow containing T. gondii. No significant differences (NS)
in anti-gluten IgG levels were found between sexes in the T. gondii-negative group. In animals that were T. gondii-positive, anti-gluten IgG levels were
significantly higher in females compared to males. Panel C: C1q antibody levels were significantly greater in T. gondii-positive vs T. gondii-negative
animals. No sex-specific differences were observed. Two-tailed t-test comparisons were used to generate listed p-values. Error bars indicate standard
errors of the mean.
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org7 November 2012 | Volume 7 | Issue 11 | e50991
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org8 November 2012 | Volume 7 | Issue 11 | e50991
the ability of other intestinally-active pathogens to generate diet-
related antibodies should be examined in animal models and in
It is well-documented that stress leads to increased intestinal
permeability [49,50,51] which, in turn, brings to question the
inflammatory state of the GI tract brought on by T. gondii
infection. It is not known with certainty how T. gondii strains gain
access to systemic circulation, but a para-cellular route affecting
epithelial tight junction proteins is suspected [52,53,54]. Following
PO infection of mice with T. gondii, parasites can be detected in the
lamina propria and in Peyer’s patches of the small intestine by one
hour post-infection, and parasites are detected in circulation by 48
hours post-infection . PO T. gondii infection is used to generate
ileitis and colonitis in mouse models, and therefore can promote
an environment of inflammation that damages cells lining the GI
tract [29,34,53,55,56]). Physical damage that compromises the GI
barrier integrity in turn promotes systemic inflammation as
intestinal bacteria are translocated into circulation . In fact,
Hand and colleagues recently established that PO infection with
T. gondii directly causes bacterial translocation of resident
commensals, loss of immune tolerance to resident microbiota
and activation of microbiota-specific T cells and inflammatory
effector cells .
If T. gondii crosses into circulation without damaging barrier
integrity, then we must speculate on other means by which the
anti-gluten immune activation originates. The para- vs trans-
cellular travel of gluten peptides across the GI barrier is itself not
well-understood, but evidence for gluten association with both
routes exists [57,58,59]. Sapone et al demonstrated that celiac
disease and gluten sensitivity were, in fact, two separate clinical
conditions based on standard GI permeability tests, which showed
that only celiac disease was associated with GI leakage . It may
be that gluten directs its own passage as evident by interactions of
gluten peptides with the intestinal permeability mediator, zonulin
and other tight junction proteins; however, gluten-associated
transcytosis has also been documented [57,58,59,61,62,63,64,65].
The anti-gluten immune activation following exposure to
T. gondii has especially interesting implications for exploring
possible neurodevelopmental mechanisms by which gluten sensi-
tivity might be involved with diseases such as schizophrenia and
autism. In these neurodevelopmental disorders, a food antigen
pathology has long been suspected for a subset of individuals, yet
direct evidence for prenatal gluten exposure and subsequent gluten
involvement in the central nervous system has been elusive. In a
large birth registry-based study in Sweden, elevated levels of
maternal anti-gluten IgG were recently found to increase the risk
of non-affective psychosis in offspring . Our experiments
demonstrate that exposure to T. gondii infection during fetal
development results in an anti-gluten immune response in
offspring. Furthermore, we document that the complement
pathway is also activated during this time period. C1q was used
here as a gauge for generalized systemic immune activation
following infection. Of interest to psychiatric diseases, however, is
the role of C1q in synaptic pruning during development [67,68].
In the early postnatal days of mouse CNS development, cortical
C1q mRNA expression is extensive compared to activity at
postnatal day 30 [67,68]. In our study, serological C1q was
significantly elevated in postnatal day 7 offspring exposed to T.
gondii compared to unexposed offspring. Future studies are
planned to examine how such prenatal exposures might affect
subsequent synaptic organization.
In summary, the models described in this paper provide
appropriate experimental tools to examine the impacts of gluten
peptides, T. gondii and associated immune activation on brain
physiology. As we accumulate more information from analyses of
clinical biomarkers, we can adapt these animal models to test the
effects of dietary modifications and other types of infections on
behavioral endpoints, the pharmacological outcomes of specific
antipsychotics on immune system parameters, and the autoim-
mune responses triggered by T. gondii infection. Ultimately, we
envision a translational system by which we can fully evaluate the
interface of environmental perturbation and genetic predisposition
as it relates to serious neurodevelopmental disorders such as
schizophrenia, bipolar disorder and autism.
We thank Dr. Lorraine Jones-Brando for a critical reading of this
manuscript. We thank Dr. Donald D. Kasarda of the U.S. Department of
Agriculture for providing us with the wheat flour used for gluten extraction.
We also thank Chunxia Yang and Maleeha Syed for technical assistance.
Conceived and designed the experiments: EGS GK MVP RHY.
Performed the experiments: GK KLG JX. Analyzed the data: EGS.
Contributed reagents/materials/analysis tools: EGS AA MVP RHY.
Wrote the paper: EGS AA MVP RHY.
1. Alaedini A, Green PH (2005) Narrative review: celiac disease: understanding a
complex autoimmune disorder. Annals of Internal Medicine 142: 289–298.
2. Dickerson F, Stallings C, Origoni A, Vaughan C, Khushalani S, et al. (2011)
Markers of gluten sensitivity and celiac disease in bipolar disorder. Bipolar
3. Dickerson F, Stallings C, Origoni A, Vaughan C, Khushalani S, et al. (2010)
Markers of gluten sensitivity and celiac disease in recent-onset psychosis and
multi-episode schizophrenia. Biol Psychiatry 68: 100–104.
4. Dohan FC (1979) Schizophrenia and neuroactive peptides from food. Lancet 1:
5. Dohan FC (1970) Coeliac disease and schizophrenia. Lancet 1: 897–898.
6. Dohan FC (1980) Hypothesis: genes and neuroactive peptides from food as cause
of schizophrenia. Adv Biochem Psychopharmacol 22: 535–548.
7. Dohan FC (1981) Schizophrenia, celiac disease, gluten antibodies, and the
importance of beta. Biol Psychiatry 16: 1115–1117.
8. Reichelt KL, Seim AR, Reichelt WH (1996) Could schizophrenia be reasonably
explained by Dohan’s hypothesis on genetic interaction with a dietary peptide
overload? Progress in Neuro-Psychopharmacology & Biological Psychiatry 20:
9. Whiteley P, Haracopos D, Knivsberg AM, Reichelt KL, Parlar S, et al. (2010)
The ScanBrit randomised, controlled, single-blind study of a gluten- and casein-
free dietary intervention for children with autism spectrum disorders. Nutr
Neurosci 13: 87–100.
10. Reichelt KL (1994) Exorphins in Schizophrenia and Autism. Journal of
Neurochemistry 63: S86–S86.
Figure 5. IgG antibody levels following prenatal exposure to T. gondii. Panel A: In 7-day old pups, anti-T. gondii IgG levels were significantly
elevated in those whose mothers were seropositive for T. gondii compared to those whose mothers were uninfected. Panel B: 7-day old offspring
exposed prenatally to T. gondii had significantly greater IgG to gluten than offspring from uninfected mothers. Panel C: 7-day old offspring of mothers
who were seropositive for T. gondii had significantly stronger complement factor C1q activation than offspring from uninfected mothers. Two-tailed
t-test comparisons were used to generate listed significant p-values. Error bars indicate standard errors of the mean.
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org9 November 2012 | Volume 7 | Issue 11 | e50991
11. Severance EG, Alaedini A, Yang S, Halling M, Gressitt KL, et al. (2012) Download full-text
Gastrointestinal inflammation and associated immune activation in schizophre-
nia. Schizophr Res 138: 48–53.
12. Severance EG, Dickerson FB, Halling M, Krivogorsky B, Haile L, et al. (2010)
Subunit and whole molecule specificity of the anti-bovine casein immune
response in recent onset psychosis and schizophrenia. Schizophr Res 118: 240–
13. Severance EG, Dupont D, Dickerson FB, Stallings CR, Origoni AE, et al. (2010)
Immune activation by casein dietary antigens in bipolar disorder. Bipolar Disord
14. Samaroo D, Dickerson F, Kasarda DD, Green PH, Briani C, et al. (2010) Novel
immune response to gluten in individuals with schizophrenia. Schizophr Res
15. Dohan FC (1988) Genetic hypothesis of idiopathic schizophrenia: its exorphin
connection. Schizophr Bull 14: 489–494.
16. Reichelt KL (1991) Peptides in Schizophrenia. Biol Psychiatry 29: 515–516.
17. Reichelt KL, Reichelt WH, Stensrud M (1995) The role of peptides in
schizophrenia. Journal of Neurochemistry 65: S43–S43.
18. Reichelt KL, Stensrud M (1998) Increase in urinary peptides prior to the
diagnosis of schizophrenia. Schizophr Res 34: 211–213.
19. Flegr J, Lenochova P, Hodny Z, Vondrova M (2011) Fatal attraction
phenomenon in humans: cat odour attractiveness increased for toxoplasma-
infected men while decreased for infected women. PLoS Neglected Tropical
Diseases 5: e1389.
20. Gatkowska J, Wieczorek M, Dziadek B, Dzitko K, Dlugonska H (2012)
Behavioral changes in mice caused by Toxoplasma gondii invasion of brain.
Parasitology Research 111: 53–8.
21. Goodwin DG, Hrubec TC, Klein BG, Strobl JS, Werre SR, et al. (2012)
Congenital infection of mice with Toxoplasma gondii induces minimal change in
behavior and no change in neurotransmitter concentrations. The Journal of
Parasitology 98: 706–12.
22. Kannan G, Moldovan K, Xiao JC, Yolken RH, Jones-Brando L, et al. (2010)
Toxoplasma gondii strain-dependent effects on mouse behaviour. Folia para-
sitologica 57: 151–155.
23. Torrey EF, Bartko JJ, Yolken RH (2012) Toxoplasma gondii and other risk factors
for schizophrenia: An update. Schizophr Bull 38: 642–7.
24. Torrey EF, Bartko JJ, Lun ZR, Yolken RH (2007) Antibodies to Toxoplasma gondii
in patients with schizophrenia: a meta-analysis. Schizophr Bull 33: 729–736.
25. Webster JP, McConkey GA (2010) Toxoplasma gondii-altered host behaviour: clues
as to mechanism of action. Folia parasitologica 57: 95–104.
26. Webster JP (2007) The effect of Toxoplasma gondii on animal behavior: playing cat
and mouse. Schizophr Bull 33: 752–756.
27. Webster JP (2001) Rats, cats, people and parasites: the impact of latent
toxoplasmosis on behaviour. Microbes and infection/Institut Pasteur 3: 1037–
28. Xiao J, Kannan G, Jones-Brando L, Brannock C, Krasnova IN, et al. (2012)
Sex-specific changes in gene expression and behavior induced by chronic
Toxoplasma infection in mice. Neuroscience 206: 39–48.
29. Bereswill S, Munoz M, Fischer A, Plickert R, Haag LM, et al. (2010) Anti-
inflammatory effects of resveratrol, curcumin and simvastatin in acute small
intestinal inflammation. PLoS One 5: e15099.
30. Erridge C, Duncan SH, Bereswill S, Heimesaat MM (2010) The induction of
colitis and ileitis in mice is associated with marked increases in intestinal
concentrations of stimulants of TLRs 2, 4, and 5. PloS One 5: e9125.
31. Munoz M, Heimesaat MM, Danker K, Struck D, Lohmann U, et al. (2009)
Interleukin (IL)-23 mediates Toxoplasma gondii-induced immunopathology in the
gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17. J Exp
Med 206: 3047–3059.
32. Schreiner M, Liesenfeld O (2009) Small intestinal inflammation following oral
infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice:
review of 70 reports from the literature. Mem Inst Oswaldo Cruz 104: 221–233.
33. Craven M, Egan CE, Dowd SE, McDonough SP, Dogan B, et al. (2012)
Inflammation drives dysbiosis and bacterial invasion in murine models of ileal
Crohn’s disease. PloS One 7: e41594.
34. Hand TW, Dos Santos LM, Bouladoux N, Molloy MJ, Pagan AJ, et al. (2012)
Acute gastrointestinal infection induces long-lived microbiota-specific T cell
responses. Science 337: 1553–1556.
35. Lidar M, Lipschitz N, Langevitz P, Barzilai O, Ram M, et al. (2009) Infectious
serologies and autoantibodies in Wegener’s granulomatosis and other vasculit-
ides: novel associations disclosed using the Rad BioPlex 2200. Annals of the New
York Academy of Sciences 1173: 649–657.
36. Rostami Nejad M, Rostami K, Cheraghipour K, Nazemalhosseini Mojarad E,
Volta U, et al. (2011) Celiac disease increases the risk of Toxoplasma gondii
infection in a large cohort of pregnant women. The American Journal of
Gastroenterology 106: 548–549.
37. Frank MM, Fries LF (1991) The role of complement in inflammation and
phagocytosis. Immunology Today 12: 322–326.
38. Barrington R, Zhang M, Fischer M, Carroll MC (2001) The role of complement
in inflammation and adaptive immunity. Immunol Rev 180: 5–15.
39. Walport MJ (2001) Complement. First of two parts. The New England Journal
of Medicine 344: 1058–1066.
40. Walport MJ (2001) Complement. Second of two parts. The New England
Journal of Medicine 344: 1140–1144.
41. Xiao J, Buka SL, Cannon TD, Suzuki Y, Viscidi RP, et al. (2009) Serological
pattern consistent with infection with type I Toxoplasma gondii in mothers and risk
of psychosis among adult offspring. Microbes Infect 11: 1011–1018.
42. Severance EG, Gressitt K, Halling M, Stallings CR, Origoni AE, et al. (2012)
Complement C1q formation of immune complexes with milk caseins and wheat
glutens in schizophrenia. Neurobiology of Disease 48: 447–53.
43. Humphrey JH, Batty I (1976) Letter: International units and standards. Lancet
44. Roberts CW, Cruickshank SM, Alexander J (1995) Sex-determined resistance to
Toxoplasma gondii is associated with temporal differences in cytokine production.
Infection and immunity 63: 2549–2555.
45. Liesenfeld O, Nguyen TA, Pharke C, Suzuki Y (2001) Importance of gender and
sex hormones in regulation of susceptibility of the small intestine to peroral
infection with Toxoplasma gondii tissue cysts. The Journal of Parasitology 87:
46. Drude S, Geissler A, Olfe J, Starke A, Domanska G, et al. (2011) Side effects of
control treatment can conceal experimental data when studying stress responses
to injection and psychological stress in mice. Lab Animal 40: 119–128.
47. Silva IA, El Nabawi M, Hoover D, Silbergeld EK (2005) Prenatal HgCl2
exposure in BALB/c mice: gender-specific effects on the ontogeny of the
immune system. Developmental and Comparative Immunology 29: 171–183.
48. Wynne O, Horvat JC, Osei-Kumah A, Smith R, Hansbro PM, et al. (2011)
Early life infection alters adult BALB/c hippocampal gene expression in a sex
specific manner. Stress 14: 247–261.
49. Lambert GP (2009) Stress-induced gastrointestinal barrier dysfunction and its
inflammatory effects. Journal of Animal Science 87: E101–108.
50. Collins SM, Bercik P (2009) The relationship between intestinal microbiota and
the central nervous system in normal gastrointestinal function and disease.
Gastroenterology 136: 2003–2014.
51. Soderholm JD, Perdue MH (2001) Stress and gastrointestinal tract. II. Stress and
intestinal barrier function. American Journal of Physiology Gastrointestinal and
Liver Physiology 280: G7–G13.
52. Munoz M, Liesenfeld O, Heimesaat MM (2011) Immunology of Toxoplasma
gondii. Immunological Reviews 240: 269–285.
53. Liesenfeld O (1999) Immune responses to Toxoplasma gondii in the gut.
Immunobiology 201: 229–239.
54. Weight CM, Carding SR (2012) The protozoan pathogen Toxoplasma gondii
targets the paracellular pathway to invade the intestinal epithelium. Annals of
the New York Academy of Sciences 1258: 135–142.
55. Liesenfeld O (2002) Oral infection of C57BL/6 mice with Toxoplasma gondii: a
new model of inflammatory bowel disease? J Infect Dis 185 Suppl 1: S96–101.
56. Egan CE, Cohen SB, Denkers EY (2011) Insights into inflammatory bowel
disease using Toxoplasma gondii as an infectious trigger. Immunology and Cell
Biology 90: 668–75.
57. Menard S, Lebreton C, Schumann M, Matysiak-Budnik T, Dugave C, et al.
(2012) Paracellular versus transcellular intestinal permeability to gliadin peptides
in active celiac disease. The American Journal of Pathology 180: 608–615.
58. Matysiak-Budnik T, Candalh C, Dugave C, Namane A, Cellier C, et al. (2003)
Alterations of the intestinal transport and processing of gliadin peptides in celiac
disease. Gastroenterology 125: 696–707.
59. Heyman M, Abed J, Lebreton C, Cerf-Bensussan N (2011) Intestinal
permeability in coeliac disease: insight into mechanisms and relevance to
pathogenesis. Gut 61: 1355–64.
60. Sapone A, Lammers KM, Casolaro V, Cammarota M, Giuliano MT, et al.
(2011) Divergence of gut permeability and mucosal immune gene expression in
two gluten-associated conditions: celiac disease and gluten sensitivity. BMC
Medicine 9: 23.
61. Lammers KM, Lu R, Brownley J, Lu B, Gerard C, et al. (2008) Gliadin induces
an increase in intestinal permeability and zonulin release by binding to the
chemokine receptor CXCR3. Gastroenterology 135: 194–204 e193.
62. Tripathi A, Lammers KM, Goldblum S, Shea-Donohue T, Netzel-Arnett S, et
al. (2009) Identification of human zonulin, a physiological modulator of tight
junctions, as prehaptoglobin-2. Proceedings of the National Academy of
Sciences of the United States of America 106: 16799–16804.
63. Drago S, El Asmar R, Di Pierro M, Grazia Clemente M, Tripathi A, et al.
(2006) Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac
intestinal mucosa and intestinal cell lines. Scandinavian Journal of Gastroen-
terology 41: 408–419.
64. Sander GR, Cummins AG, Henshall T, Powell BC (2005) Rapid disruption of
intestinal barrier function by gliadin involves altered expression of apical
junctional proteins. FEBS Letters 579: 4851–4855.
65. Rallabhandi P (2012) Gluten and celiac disease–an immunological perspective.
Journal of AOAC International 95: 349–355.
66. Karlsson H, Blomstrom A, Wicks S, Yang S, Yolken RH, et al. (2012) Maternal
Antibodies to Dietary Antigens and Risk for Nonaffective Psychosis in Offspring.
The American Journal of Psychiatry 169: 625–32.
67. Boulanger LM (2009) Immune proteins in brain development and synaptic
plasticity. Neuron 64: 93–109.
68. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, et al. (2007)
The classical complement cascade mediates CNS synapse elimination. Cell 131:
Anti-Gluten Immune Response
PLOS ONE | www.plosone.org10 November 2012 | Volume 7 | Issue 11 | e50991