pnas201202556 12776..12781 - PNAS-2012-Hsiao-12776-81.pdf

Modeling an autism risk factor in mice leads to

permanent immune dysregulation

Elaine Y. Hsiao

, Sara W. McBride, Janet Chow, Sarkis K. Mazmanian, and Paul H. Patterson

Biology Division, California Institute of Technology, Pasadena, CA 91125

Edited by Carla J. Shatz, Stanford University, Stanford, CA, and approved June 19, 2012 (received for review February 15, 2012)

Increasing evidence highlights a role for the immune system in the

pathogenesis of autism spectrum disorder (ASD), as immune

dysregulation is observed in the brain, periphery, and gastroin-

testinal tract of ASD individuals. Furthermore, maternal infection

(maternal immune activation, MIA) is a risk factor for ASD.

Modeling this risk factor in mice yields offspring with the cardinal

behavioral and neuropathological symptoms of human ASD. In

this study, we

fi

nd that offspring of immune-activated mothers

display altered immune pro

fi

les and function, characterized by

a systemic de

fi

cit in CD4

TCR

β

Foxp3

CD25

T regulatory cells,

increased IL-6 and IL-17 production by CD4

T cells, and elevated

levels of peripheral Gr-1

cells. In addition, hematopoietic stem

cells from MIA offspring exhibit altered myeloid lineage potential

and differentiation. Interestingly, repopulating irradiated control

mice with bone marrow derived from MIA offspring does not con-

fer MIA-related immunological de

fi

cits, implicating the peripheral

environmental context in long-term programming of immune dys-

function. Furthermore, behaviorally abnormal MIA offspring that

have been irradiated and transplanted with immunologically nor-

mal bone marrow from either MIA or control offspring no longer

exhibit de

fi

cits in stereotyped/repetitive and anxiety-like behav-

iors, suggesting that immune abnormalities in MIA offspring

can contribute to ASD-related behaviors. These studies support

a link between cellular immune dysregulation and ASD-related

behavioral de

fi

cits in a mouse model of an autism risk factor.

immunity

|

neurodevelopment

|

prenatal programming

|

neuroimmunology

utism is a complex neurodevelopmental disorder and a

pressing medical concern, affecting over 1% of children in

the United States (1). Although autism spectrum disorder (ASD)

is characterized by stereotypic behaviors and language and social

cits, increasing evidence suggests a role for the immune

system in ASD pathogenesis. Altered cytokine pro

les in the

postmortem brain, cerebrospinal

uid, and plasma are found in

ASD, and several studies have demonstrated an elevated number

and activation of microglia and astrocytes in the postmortem

brain (2). There are also many reports of peripheral immune

abnormalities in autistic individuals, including increased NK cell

activity, differential monocyte responses to in vitro stimulation,

and altered serum Ig levels (3, 4).

Abnormal activation of the immune system may also be in-

volved in the etiology of autism. Several studies have associated

ASD risk with immune-related susceptibility genes, such as those

encoding MET receptor tyrosine kinase, PRKCB1, complement

C4B, and speci

c HLA haplotypes (3, 4). In addition, antibrain

antibodies are elevated in some ASD sera and in some mothers of

autistic children (5

–

7). Family members of autistic children, par-

ticularly the mothers, show a higher incidence of allergy or auto-

immune diseases (8, 9). Consistent with immune involvement are

ndings that maternal infection is a risk factor for autism (2).

After the 1964 rubella pandemic, 8

–

13% of children born to

infected mothers developed features of autism (10). In a recent

study surveying all children born in Denmark from 1980 to 2005,

averysigni

cant association was found between autism and ma-

ternal viral infection during the

rst trimester of pregnancy (11).

Moreover, elevation of IFN-

, IL-4, or IL-5 in maternal serum is

associated with increased risk for ASD in the offspring (12), as is

elevation of monocyte chemoattractant protein-1 in the amniotic

uid (13).

Whether the immune abnormalities in ASD actually contribute

to its behavioral symptoms or whether they are an epiphenome-

non of primary neural dysfunction is an outstanding question. The

immune system exhibits lifelong reciprocal interactions with the

central nervous system, and the fact that the immune status can

uence behavioral responses is exempli

ed by early studies

demonstrating that responses to infection and in

ammation are

relayed to the brain, resulting in the induction of fever and sick-

ness behavior (14). Moreover, administration of certain cytokines

to human subjects frequently causes striking changes in mental

state (15). Conversely, emotional and psychological state can in-

uence immune function. Perhaps the strongest evidence to date

is the

nding that both short- and long-term stress lead to dis-

ruption of immune function (16). Immune dysregulation has also

been implicated in the etiology of a variety of neurodegenerative,

psychiatric, and neurodevelopmental disorders, including Parkin-

son, Huntington, and Alzheimer

’

s diseases, multiple sclerosis,

major depression, schizophrenia, and addiction (17

–

20).

Here we ask whether a mouse model exhibiting many features

of autism also displays altered immune function. We use the

maternal immune activation (MIA) model, which is based on

maternal infection as a key environmental risk factor for autism.

Pregnant mice are injected with the synthetic, double-stranded

RNA, poly(I:C), to initiate a proin

ammatory antiviral response.

This type of MIA yields offspring with the core behavioral and

neuropathological symptoms of autism, including reduced social

interaction, abnormal communication, stereotyped/repetitive be-

havior, and a spatially restricted de

cit in Purkinje cells (21, 22).

In this study, we pro

le peripheral immune subtypes, assess

the functional activities of major leukocyte lineages and evaluate

the lineage potential of fetal and adult hematopoietic stem cells

(HSCs) and progenitors. To explore the potential for prenatal

programming of long-term immune dysfunction, we examine

whether transferring HSCs from MIA offspring into non-MIA

offspring can induce cell-autonomous immune abnormalities. To

gain insight into whether immune abnormalities in MIA off-

spring contribute to the pathogenesis of ASD-related behaviors,

we behaviorally assess poly(I:C) offspring repopulated with bone

marrow (BM) from saline offspring. Our

ndings demonstrate

that immune challenge during prenatal life leads to persistent

immune alterations in the postnatal offspring, which can further

impact the development or maintenance of abnormal behavior.

Results

MIA Offspring Exhibit De

fi

cits in Regulatory T Cells and Elevated CD4

T-Cell Responses.

Many studies of immune dysregulation in human

ASD report a bias toward a proin

ammatory phenotype (3, 4).

Author contributions: E.Y.H., S.W.M., J.C., S.K.M., and P.H.P. designed research; E.Y.H.,

S.W.M., and J.C. performed research; E.Y.H., S.W.M., J.C., and P.H.P. analyzed data; and

E.Y.H., S.W.M., J.C., S.K.M., and P.H.P. wrote the paper.

The authors declare no con

fl

ict of interest.

This article is a PNAS Direct Submission.

To whom correspondence should be addressed. E-mail: ehsiao@caltech.edu.

This article contains supporting information online at

www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1202556109/-/DCSupplemental

12776

–

12781

PNAS

July 31, 2012

vol. 109

no. 31

www.pnas.org/cgi/doi/10.1073/pnas.1202556109

We therefore investigated regulatory T cells (Tregs) as known

suppressors of the innate and adaptive immune responses.

Compared with controls, adult offspring of poly(I:C)-injected

mothers display a

∼

50% decrease in splenic CD4

Foxp3

CD25

Tregs (Fig. 1

). This de

cit is also re

ected in signi

cantly decreased levels of total CD4

Foxp3

cells and possible

decreases (

= 0.0846) in CD4

Foxp3

CD25

−

T cells. Similar

cits in total CD4

Foxp3

, Foxp3

CD25

−

, and Foxp3

CD25

cells are also observed in mesenteric lymph nodes

(MLNs) from adult poly(I:C) offspring (

Fig. S1

). Despite these

differences in Treg levels, there is no difference in the suppres-

sion of CD4

CD25

−

T-cell proliferation between Tregs from

saline versus poly(I:C) offspring (

Fig. S2

). Taken together, these

ndings indicate a systemic de

cit in the abundance of Tregs.

We further tested MIA offspring for levels of IL-17

–

producing

CD4

T (Th17) cells, given their proin

ammatory nature and

reported reciprocal relationship with Tregs. In assays done in

parallel with those for Tregs, we

nd no signi

cant difference

from controls in levels of CD4

TCR

IL-17

(Th17) cells,

with or without IFN-

expression (Fig. 1

). There is also no

difference in the level of CD4

TCR

IL-17

−

IFN-

(Th1)

cells. To further examine CD4

T cells, we measured their se-

cretion of IL-6 and IL-17 in response to in vitro stimulation.

Compared with controls, CD4

cells from spleens of 15-wk-old

poly(I:C) offspring release signi

cantly more IL-6 and IL-17

after in vitro stimulation, with no difference in TNF-

secretion

(Fig. 1

). This result is similarly observed with splenic CD4

T cells derived from 3-wk-old offspring (

Fig. S3

) and 1-y-old

offspring (

Fig. S3

), suggesting an early onset of persistent im-

mune dysfunction. Interestingly, splenic CD4

T cells from 3-wk-

old mice produce much lower levels of IL-17 than do such cells

from adult offspring (0

–

10 pg/mL compared with 50

–

400 pg/

mL), re

ecting the immunological immaturity reported in young

versus adult mice and humans (23). CD4

T cells from MLNs of

poly(I:C) offspring are also hyperresponsive to in vitro stimula-

tion, suggesting that this abnormality is common to secondary

lymphoid organs (

Fig. S1

and

). Overall, MIA leads to lower

Treg levels and elevated CD4

T-cell responsiveness in spleens

and MLNs from MIA offspring. This result can be characterized

as a persistent, proin

ammatory T-helper-cell phenotype.

MIA Offspring Display Increased Levels of Gr-1

Cells and Skewed HSC

Differentiation.

To determine whether MIA during fetal de-

velopment alters the pro

le of other immune subtypes in the

offspring, we assessed major leukocyte classes in spleens from

poly(I:C) and saline offspring. Compared with controls, adult

poly(I:C) offspring exhibit a 1.5-fold higher level of Gr-1

cells

and trending increase in CD11b

cells (

= 0.1256) (Fig. 2

). In

contrast, there is no difference from controls in the percentages

of total B220

B cells, NK1.1

NK cells, CD4

T cells, or CD8

T cells. Moreover, no signi

cant differences are detected for any

of the primary leukocyte subtypes in the MLNs (

Fig. S1

Gr-1

cells re

ect a heterogeneous group of immune subtypes

that includes neutrophils and monocytes, in

ammatory cells, and

suppressor cells, alike. To determine whether the elevated levels

of Gr-1

cells observed in poly(I:C) spleens could be attributed

to particular Gr-1

subtypes, we further characterized the

splenic Gr-1

population using CD11b, Ly6C, and Ly6G mark-

ers. Interestingly, poly(I:C) offspring exhibit mild increases in all

three populations of Gr-1

cells resolved: (

) Gr-1

CD11b

Ly6C

mid

Ly6G

SSC

mid

) Gr-1

mid

CD11b

Ly6C

mid

Ly6G

mid

SSC

mid

, and (

iii

) Gr-1

mid

CD11b

Ly6C

Ly6G- SSC

(

Fig. S4

Gr-1

subtypes

and

are referred to as neutrophils, by his-

tology and in line with their high granularity, and subtype

iii

identi

ed as a monocyte population (24). Overall, that poly(I:C)

offspring display increases in all identi

ed Gr-1

populations

suggests that skewing at the HSC or progenitor level may un-

derlie the elevated Gr-1 phenotype observed in adult poly(I:C)

offspring compared with controls.

To evaluate the origin of the increase in splenic Gr-1

cells,

we assessed the lineage potential and differentiation of BM cells

from adult poly(I:C) versus saline offspring. Using a colony-

forming assay to morphologically assess lineage differentiation,

nd that HSCs and progenitors from poly(I:C) offspring BM

exhibit increased differentiation into CFU-G (granulocyte) pre-

cursors and decreased differentiation into early CFU-GM

(granulocyte-macrophage) precursors (Fig. 2

). Turning to the

fetus, we

nd that similar differentiation is observed with fetal

liver HSCs and progenitors from poly(I:C) offspring (Fig. 2

Thus, MIA induces preferential differentiation of fetal as well as

adult HSCs and progenitors into granulocyte precursors, which

may account for the increased levels of mature Gr-1

cells in

spleens of poly(I:C) offspring compared with controls.

Immune Abnormalities Observed in MIA Offspring Are Not Transferred

via BM Transplant.

Our data demonstrate that MIA leads to an

altered pro

le of peripheral immune cells in the offspring,

characterized by decreased levels of Tregs, hyperresponsive

CD4

T cells, and elevated levels of Gr-1

cells. To explore

whether these immune abnormalities (and the Gr-1 phenotype,

in particular) can be attributed to cell-intrinsic developmental

programming of HSCs, we transferred BM from the immuno-

logically aberrant poly(I:C) offspring into irradiated saline and

Fig. 1.

MIA leads to decreased levels of

Tregs and hyperresponsiveness in CD4

Tcells

from spleens of adult offspring. (

) Com-

pared with controls, adult poly(I:C) offspring

exhibit decreased levels of CD4

Foxp3

splenocytes and CD4

Foxp3

CD25

Tregs

(

= 5, where each sample represents a pool

of three spleens). (

) Adult poly(I:C) offspring

exhibit decreased levels of splenic CD4

TCR

Foxp3

CD25

Tregs, but no signi

fi

cant

difference in splenic CD4

TCR

IFN-

IL-17

Th17

Th1 cells or CD4

TCR

IFN-

−

IL-17

Th17 cells (

= 4, where each sample repre-

sents a pool of three spleens). (

)CD4

Tcells

from the spleens of adult offspring secrete

elevated levels of IL-6 and IL-17 in response

to PMA/ionomycin stimulation. In contrast,

CD4

T cells from spleens of adult poly(I:C)

and saline offspring do not differ the level

of TNF-

secreted (

=11

–

16). *

0.05,

0.01, ***

0.001; n.s., not signi

fi

cant.

All panels show one representative experi-

ment of at least two separate trials.

Hsiao et al.

PNAS

July 31, 2012

vol. 109

no. 31

12777

NEUROSCIENCE

poly(I:C) offspring and assessed whether any MIA-associated

immune abnormalities were reestablished.

nd that saline and poly(I:C) offspring that were irradi-

ated and reconstituted with poly(I:C) BM do not develop or

retain any of the immune abnormalities exhibited by un-

manipulated MIA offspring. All BM-transplanted groups exhibit

levels of splenic CD4

TCR

CD25

Foxp3

Tregs and Gr-1

cells that are comparable to those observed in untransplanted

saline offspring (Fig. 3

and

). There is also no difference

between saline and BM-transplanted mice in distributions of

progenitor subtypes after in vitro differentiation of BM HSCs

(

Fig. S5

). Furthermore, CD4

T cells isolated from spleens of

any of the types of BM-transplanted mice secrete levels of IL-6

and IL-17 in response to phorbol 12-myristate 13-acetate

(PMA)/ionomycin stimulation that are similar to those produced

by CD4

T cells isolated from spleens of untransplanted saline

offspring (Fig. 3

). Importantly, the fact that saline mice trans-

planted with saline BM display equivalent levels of Tregs and

Gr-1

cells and similar CD4

T-cell responsiveness to those

observed in unmanipulated saline offspring suggests that the ir-

radiation and BM-transplant procedure itself does not skew

immune system pro

les under these conditions. Overall, these

experiments indicate that MIA-associated immune abnormalities

are not transferred via transplant of poly(I:C) BM into irradiated

saline or poly(I:C) offspring. This

nding suggests either that the

HSC microenvironment is important for maintaining altered

HSC potential in MIA offspring, or that the particular MIA-

associated immune abnormality is not programmed at the stem

cell level.

BM Transplant in MIA Offspring Normalizes Repetitive and Anxiety-

Like Behavioral Abnormalities.

To investigate whether the immune

abnormalities found in MIA offspring are an independent par-

allel pathology or if they actually contribute to the development

or maintenance of ASD-like behaviors, we assessed behavioral

performance in poly(I:C) offspring transplanted with saline BM.

Poly(I:C) offspring were

rst con

rmed to exhibit their expected

behavioral phenotypes before the procedure. Compared with

controls, poly(I:C) offspring display a de

cit in prepulse in-

hibition (PPI) (

Fig. S6

). PPI is used to measure sensorimotor

gating of the startle re

ex, and decreased PPI is frequently ob-

served in autistic individuals (25). Poly(I:C) offspring also exhibit

core behavioral symptoms of autism, including increased re-

petitive behavior as measured by higher levels of stereotypic

marble burying and decreased social preference, as assessed by

reduced duration spent and entries into a chamber housing

a novel mouse versus a familiar mouse (

Fig. S6

and

). In

addition, MIA offspring exhibit increased anxiety, as re

ected by

Fig. 2.

MIA leads to increased levels of splenic Gr-1

cells

in adult offspring and preferen

tial differentiation of HSCs

into granulocyte precursors in fetal and adult offspring.

(

) Spleens from adult poly(I:C) offspring exhibit in-

creased levels of Gr-1

cells and no signi

fi

cant differences

in other major lineages co

mpared with controls (

=4,

where each sample represent

s a pool of three spleens). (

)

Compared with BM HSCs from control offspring, BM HSCs

from adult poly(I:C) offspring display increased differen-

tiationintoCFU-Gprecursors

and decreased

differentia-

tion into early CFU-GM precursors (

=4).Comparedwith

controls, fetal liver HSCs

from embryonic day (E) 13.5 (

Left

) and E15.5 (

Right

) poly(I:C) offspring also display

increased differentiation into CFU-G and decreased dif-

ferentiation into CFU-GM, in addition to decreased per-

centages of CFU-GEMM with

E13.5 HSCs and increased

percentages of CFU-E with E15.5 HSCs (

= 4, where each

sample represents a pool of cells from six fetal livers from

a single litter). *

0.05, **

0.01, ***

0.001; n.s.,

not signi

fi

cant. All panels represent one representative

experiment of at least two separate trials.

Fig. 3.

Immune abnormalities observed in MIA offspring

are not transferred by BM transplant into irradiated mice.

(

) There is no difference in levels of splenic CD4

TCR

CD25

Foxp3

Tregs between saline or poly(I:C) offspring

transplanted with BM from saline or poly(I:C) offspring.

Levels of splenic Tregs between BM transplant groups are

comparable to those observed in untransplanted saline

offspring; untransplanted poly(I:C) offspring display a sig-

fi

cant de

fi

cit in Treg percentages (

–

5). (

) BM-

transplanted mice do not display MIA-associated increases

in splenic Gr-1

cells. Percentages of Gr-1

cells are com-

parable between BM transplanted groups and untrans-

planted saline offspring (

–

5), but untransplanted poly

(I:C) offspring exhibit signi

fi

cantly elevated levels of splenic

Gr-1

cells. (

) CD4

T cells from BM-transplanted mice

exhibit statistically equivalent levels of IL-6 (

Left

) and IL-17

(

Right

) in response to PMA/ionomycin stimulation in vitro.

There are no differences between concentrations of IL-6

and IL-17 secreted by CD4

cells from BM-transplanted

groups and from untransplanted saline offspring, despite

signi

fi

cant hyperresponsiveness of CD4

T cells from poly(I:

C) offspring [

=11

–

16 for saline and poly(I:C) groups, 4

–

for BM transplant groups]. *

0.05, **

0.01. BM

transplant data were acquired from one large experiment.

12778

www.pnas.org/cgi/doi/10.1073/pnas.1202556109

Hsiao et al.

reduced duration spent, and entries into, the center arena of an

open

eld, despite no signi

cant difference in total distance

traveled (

Fig. S6

After this initial behavioral testing, the mice were irradiated

and transplanted with donor BM harvested from either adult

saline or poly(I:C) offspring. Notably, mouse heads were shiel-

ded during the procedure to limit known effects of irradiation on

neurogenesis and glial activation, and to preclude any associated

downstream in

uences on behavioral performance. Further-

more, MIA offspring do not exhibit global changes in levels of

activated or total brain macrophages/microglia (

Fig. S7

), sug-

gesting that microglial abnormalities do not contribute the

persistence of ASD-related behaviors in these mice.

After irradiation and transplantation with saline BM, MIA

offspring no longer exhibit behavioral abnormalities in several of

these tests. Irradiation and saline BM transplant in poly(I:C)

offspring restores repetitive marble burying to levels observed in

saline controls (Fig. 4

). In addition, irradiated and BM-trans-

planted MIA offspring exhibit signi

cantly decreased anxiety-like

behavior, as measured by elevated duration of time spent

and entries into the center arena of the open

eld (Fig. 4

Interestingly, both saline and poly(I:C) BM-transplanted mice

exhibit signi

cantly increased number of center entries compared

with untransplanted offspring. This

nding is similarly re

ected by

a statistically signi

cant increase in total distance traveled by

transplanted offspring versus untransplanted controls. This

nding

may be a result of effects of the irradiation and transplant pro-

cedure on behavior rather than habituation of the mice to re-

peated testing, because we are able to replicate open

eld de

cits

of equivalent intensity in poly(I:C) offspring after retesting at

different adult ages (

Fig. S8

). On the other hand, treatment of

poly(I:C) offspring with saline BM has no signi

cant effect on the

cit in social preference, as measured by duration and number

of entries into the social chamber (Fig. 4

Although treatment of MIA offspring with irradiation and

saline BM transplantation signi

cantly restores normal behavior

in several tests, we

nd that treatment of MIA offspring with

irradiation and poly(I:C) BM transplantation also improves

those behaviors. Notably, irradiated MIA offspring transplanted

with poly(I:C) BM exhibit improved open

eld and marble-

burying performance (

Fig. S9

). This result is consistent with our

nding that transplant of poly(I:C) BM does not give rise to cell-

autonomous immune abnormalities (Fig. 3). As such, both poly

(I:C) BM and saline BM can be considered immunologically

normal in these experiments. Taken together, these data dem-

onstrate that MIA offspring that had previously exhibited ab-

normal behaviors fail to exhibit repetitive and anxiety-like

behavior after exposure to irradiation and BM transplant.

Discussion

In the present study we identify differences in dynamic cellular

immune responses in a mouse model of an autism risk factor.

Offspring of immune-activated mothers develop altered immune

pro

les and function in the spleen and MLN, which are consis-

tent with a proin

ammatory phenotype. In addition, we dem-

onstrate preferential myeloid differentiation of fetal HSCs and

progenitors, which is also found in adult HSCs. This

nding

could form the basis for the altered immune distributions in

secondary lymphoid organs from MIA offspring. However, BM

transplant of HSCs derived from MIA offspring is not suf

cient

to recapitulate the elevated levels of Gr-1

cells exhibited by

untransplanted MIA offspring. This result highlights the impor-

tance of appropriate environmental cues for preserving this

phenotype, as discussed below. We also used the irradiation-BM

transplant approach to explore the role of peripheral immune

dysfunction on behavioral performance. Interestingly, this pro-

cedure corrects some of the ASD-like behavioral symptoms in

the MIA offspring.

MIA leads to permanently hyperresponsive CD4

T cells, as

well as decreased Tregs in the offspring, suggesting a chronic,

proin

ammatory phenotype. This

nding is consistent with de-

creased numbers CD4

CD25

and CD3

GITR

T cells ob-

served in children with ASD (26, 27). Diminished Treg levels and

associated decreases in immune regulation may re

ect the

nding that autistic individuals exhibit decreased levels of regu-

latory cytokines, such as TGF-

1, and increased levels of

proin

ammatory cytokines in serum, cerebrospinal

uid and

postmortem brain (2). Furthermore, given that Tregs are critical

for limiting immune activation and preventing self-reactivity,

their de

ciency may underlie the reports of a link between ASD

and autoimmune disease (8, 28).

Our

nding that CD4

T cells from MIA offspring are hyper-

responsive to in vitro stimulation further re

ects diminished

immune homeostasis. Increases in activated DR

T cells are ob-

served in human autism (29, 30). In addition, elevated levels of

TNF-

–

and IFN-

–

producing T cells are found in peripheral

blood and gastrointestinal mucosa from autistic individuals (31). A

number of other altered immune responses have been reported in

ASD (3, 4), and we

nd that MIA offspring also exhibit some of

these changes, including altered leukocyte subsets and CD4

T-cell responses to stimulation. This overlap between

ndings of

immune dysregulation in ASD and our current results lends sup-

port to MIA as a mouse model with construct and face validity for

this disorder.

We further demonstrate that MIA during fetal development

leads to signi

cantly increased levels of peripheral Gr-1

CD11b

neutrophilic and monocytic cells in adult offspring. Granulocytosis

Fig. 4.

MIA offspring irradiated and transplanted with

saline BM exhibit decreased repetitive and anxiety-like

behavior. (

) Irradiation and transplantation of saline BM

into MIA offspring restores repetitive marble-burying be-

havior to a level at or below that observed in controls. (

)

Irradiation and transplantation of saline BM into MIA off-

spring decreases anxiety-like behavior as measured by sig-

fi

cant increases in the number of center entries (

Center

)

and duration in the center arena (

Left

), compared with

those observed in untransplanted MIA mice. Both saline

and poly(I:C) BM-transplant mice also exhibit increased

overall activity in the open

fi

eld, as measured by signi

fi

cantly increased total distance traveled compared with

untransplanted mice (

Right

). (

) Transplanting saline off-

spring BM into poly(I:C) offspring has no signi

fi

cant effect

on their social preference behavior, as measured by cham-

ber duration (

Left

). There is, however, a trending im-

provement in social preference behavior as measured by

chamber frequency in BM-transplanted MIA offspring

(

Right

). *

0.05, **

0.01; n.s., not signi

fi

cant. BM-

transplant data were acquired from one large experiment.

Hsiao et al.

PNAS

July 31, 2012

vol. 109

no. 31

12779

NEUROSCIENCE

is typically observed after acute in

ammation (32, 33) and is also

a feature of a number of chronic diseases (34

–

36). There have

been no reports of altered neutrophil levels in ASD, however. In

one study, children with ASD exhibited increased plasma mono-

cyte counts, but no signi

cant abnormalities in major granulocyte

subtypes (37). In light of the present results, it will be of interest

to extend these immunophenotypic studies to behavioral or

symptomatic subpopulations of ASD individuals.

It is intriguing to consider that MIA effects on developing

HSCs may underlie some of the persistent peripheral immune

changes found in the offspring. We

nd that both adult and fetal

HSCs and progenitor cells display preferential differentiation

into CFU-G colonies, which provides an explanation for how

increases in such short-lived cells could be maintained over the

lifespan of MIA offspring. Maternal poly(I:C) injection is known

to induce dramatic increases in proin

ammatory cytokines in the

placenta, a principal HSC niche during midgestation (38). Be-

cause HSCs and progenitors respond to in

ammatory signals,

such as cytokines and Toll-like receptor ligands, it will be in-

teresting to explore whether MIA-induced changes in the HSC

environment can skew hematopoietic lineage decisions and fate.

Despite the fact that HSCs and progenitors isolated from poly

(I:C) offspring are developmentally skewed toward granulocyte

precursors, transplantation of BM from poly(I:C) offspring into

irradiated saline offspring or back into irradiated poly(I:C) off-

spring does not transfer the Gr-1 phenotype. This

nding sug-

gests that developmental programming of HSCs to skew lineage

differentiation in MIA offspring is not governed solely by stably

encoded cell-intrinsic factors, such as epigenetic modi

cation.

Rather, it is likely that an MIA-induced peripheral environment

is necessary to supply the cues that guide preferential myeloid

development. Changes in G-CSF, IL-5, and IL-3 levels, for ex-

ample, are implicated in

ne-tuning the transcription factor ac-

tivity that governs lineage choice for granulocyte/macrophage

progenitors (39). Indeed, we have found that MIA offspring

exhibit dynamic and chronically altered peripheral blood and

splenic cytokine pro

les (40). It will be of interest to assess

whether the BM microenvironment differs in poly(I:C) and sa-

line offspring. Overall, it is intriguing that the Gr-1 phenotype

observed in MIA offspring is not transferred via BM transplant,

suggesting a lack of suf

cient cell-intrinsic epigenetic pro-

gramming and a key role for environmental factors in promoting

this lineage choice.

We demonstrate that subjecting MIA offspring that have

validated behavioral de

cits to irradiation and reconstitution

with immunologically normal BM alters their behavioral phe-

notype. Namely, these MIA offspring no longer exhibit their

previous abnormalities in repetitive marble burying and open-

eld exploration. These

ndings imply that correcting immune

function can correct some autism-related behavioral abnormal-

ities. However, this experiment has a signi

cant limitation. In the

absence of a positive control (MIA offspring that retain behav-

ioral de

cits after irradiation and BM transplantation), we are

unable to distinguish between the potential restorative effects of

the BM itself (that is, the effects of restoring immune phenotype)

versus the effect of confounding factors associated with the

transplant procedure. The most obvious of these is the effect of

irradiation on various aspects of recipient homeostasis, including

metabolic function, gastrointestinal microbial composition, and

oxidative stress, all of which may indirectly in

uence behavioral

outcome. We are, however, inspired by recent work demon-

strating that stereotyped grooming behavior in Hoxb8 mutant

mice is, in fact, transferrable via BM transplant (41). This

nding

shows, at least in that experimental model, that mice can re-

constitute an expected behavioral impairment via BM transplant

despite prior irradiation. It will be important to explore other

experimental approaches to treat immune dysfunction in MIA

offspring and to further assess whether these can alleviate

behavioral outcomes.

Nonetheless, our

nding that behavioral abnormalities in the

MIA offspring can be corrected by the irradiation-BM transplant

procedure contributes to a growing number of studies reporting

the ef

cacy of BM transplant on ameliorating symptoms of

neurological disorders (41

–

43). Furthermore, several experi-

ments using RAG1 KO, SCID, and athymic mice demonstrate

that primary immune dysfunction can lead to behavioral im-

pairment (44

–

47). Interestingly, work with germ-free mice links

the absence of microbiota and associated alterations in the im-

mune system to abnormal behavioral performance (48). Some

immune alterations have been described in the maternal valproic

acid model and the BTBR mouse strain that display behavioral

features of autism (49, 50). In contrast, much remains to be

learned about potential immune changes in mouse models of

ASD candidate genes. One such gene is particularly attractive in

this regard,

MET

, which encodes a tyrosine kinase receptor and

is known to play a role in immune regulation (51).

Altered peripheral immune pro

les and cellular activity are

also associated with core behavioral impairments in human ASD.

Increased plasma IL-4 levels correlate with greater de

cits in

communication, and increased plasma IL-8, IL-12p40, IL-6, and

IL-1

are linked to stereotypy, hyperactivity, and lethargy scores

from ASD children (52). Altered levels of other immune factors,

including TGF, macrophage migration inhibitory factor (MIF),

and CD31, have also been associated with the severity of ASD-

related behaviors or pathophysiology (4). In addition, reduced

levels of plasma IgG and IgM are associated with behavioral

severity in ASD children (53).

It is striking that in a mouse model of an autism environmental

risk factor that exhibits the cardinal behavioral and neuropatho-

logical symptoms of autism, there is also permanent peripheral

immune dysregulation. This

nding provides the opportunity to

explore molecular mechanisms underlying the relationship be-

tween brain dysfunction and altered immunity in the manifestation

of abnormal behavior. Furthermore, this

nding provides a plat-

form for investigating how prenatal challenges can program

long-term postnatal immunity, health, and disease. Maternal in-

sult-mediated epigenetic modi

cation in HSC and progenitor cells

is one possible mechanism for how effects may be established by

transient environmental changes yet persist permanently into

adulthood. However, the BM transplant results suggest that the

peripheral environment of the MIA offspring is also critical for

maintaining a permanently modi

ed immune state.

Methods

Detailed methods are provided in

SI Methods

MIA.

PregnantC57BL/6Nmicewere injected on E12.5 with saline or poly(I:C).For

poly(I:C) injections, poly(I:C) potassium salt (Sigma Aldrich) was dissolved in

saline at 4 mg/mL and administered intraperitoneally at 20 mg/kg [based on the

weight of the poly(I:C) itself, not including the total weight of the potassium

salt]. Control mice were injected with saline alone at 5

L/g body weight.

CD4

T-Cell in Vitro Stimulation.

CD4

T cells were cultured in complete

RPMI with PMA (50 ng/mL) and ionomycin (750 ng/mL) for 3 d at 37 °C with

5% (vol/vol) CO

. Each day, 0.5 mL supernatant was collected. ELISA to de-

tect IL-6, IL-17, and TNF-

were performed according to the manufacturer

’

instructions (eBioscience).

Flow Cytometry.

For subtyping of Gr-1

splenocytes, cells were stained with

Gr-1-APC, CD11b-PE, Ly6G-APC, Ly6C-FITC, and Ter119-PerCP-Cy5.5 (Biol-

egend). For detection of Th17 cells and Tregs, splenocytes were stimulated

for 4 h with PMA/ionomycin in the presence of GolgiPLUG (BD Biosciences).

Suspensions were blocked for Fc receptors and labeled with CD4-FITC, TCRb-

PerCP-Cy5.5, and CD25-PE before labeling with IFN-

–

PE and Foxp3-APC

(eBioscience). Samples were processed using the FACSCalibur cytometer (BD

Biosciences). Data were analyzed using FlowJo software (TreeStar).

Methylcellulose Colony-Forming Assay.

fetal liver or BM cells were

added to 3 mL of thawed methylcellulose supplemented with SCF, IL-3, IL-6,

and Epo (StemCell). Total colony number was counted on day 5 and con

fi

rmed

to be equivalent across groups. CFU-G, -M, -E (erythroid), -GM, and -GEMM

colonies were blindly scored on day 12 according to standard protocols.

12780

www.pnas.org/cgi/doi/10.1073/pnas.1202556109

Hsiao et al.

BM Transplantation.

Behaviorally tested, 9-wk-old mice were injected in-

traperitoneally with high-dose ketamine/xylazine (5.6

L/1 g per mouse) and

lethally irradiated (1,000 rads) with heads shielded by 4-mm lead. The ef-

fectiveness of the head shields was con

fi

rmed by selective graying of black

coat color in the irradiated area. Recovered mice were anesthetized with

iso

fl

uorane and injected retro-orbitally with 5

donor BM cells. Mice

were retested in behavioral paradigms at 17

–

19 wk of age and killed for

immunological assays at 19

–

20 wk of age.

Behavioral Testing.

At 6

–

8 wk of age, mice were behaviorally tested for PPI,

open-

fi

eld exploration, repetitive marble burying, and social preference (22,

38, 54, 55). Eight weeks after transplant, mice were similarly tested in all

paradigms but PPI because PPI performance is highly sensitive to handling

and prior testing experience (56).

Statistical Analysis.

Statistical analysis was performed with Prism software

(Graphpad). Differences between two treatment groups were assessed using

Student

test, and differences among multiple groups were assessed using

one-way ANOVA and Bonferroni post hoc test. Two-way ANOVA and Bon-

ferroni post hoc test was used for PPI, CD4

T-cell stimulation and methyl-

cellulose assay data.

ACKNOWLEDGMENTS.

The authors acknowledge the kind assistance of

B. Deverman and G. Sharon for reviewing the manuscript, R. Sauza for caring

for the animals, and M. Sadoshima for technical help. This work was

supported by an Autism Speaks Weatherstone Fellowship (to E.Y.H.);

National Institutes of Health Graduate Training Grant NIH/NRSA 5 T32

GM07737 (to E.Y.H. and J.C.); a Weston Havens Foundation grant (to S.K.M.

and P.H.P.); a Caltech Innovation grant (to S.K.M. and P.H.P.); and a

Congressionally Directed Medical Research Program Idea Development

Award (to S.K.M. and P.H.P.).

1. Autism and Developmental Disabilities Monitoring Network Surveillance Year 2008

Principal Investigators; Centers for Disease Control and Prevention (2012) Prevalence

of autism spectrum disorders

—

Autism and Developmental Disabilities Monitoring

Network, 14 sites, United States, 2008.

MMWR Surveill Summ

61:1

–

19.

2. Patterson PH (2011) Maternal infection and immune involvement in autism.

Trends

Mol Med

17:389

–

394.

3. Goines P, Van de Water J (2010) The immune system

’

s role in the biology of autism.

Curr Opin Neurol

23:111

–

117.

4. Onore C, Careaga M, Ashwood P (2012) The role of immune dysfunction in the

pathophysiology of autism.

Brain Behav Immun

26:383

–

392.

5. Croen LA, et al. (2008) Maternal mid-pregnancy autoantibodies to fetal brain protein:

The early markers for autism study.

Biol Psychiatry

64:583

–

588.

6. Singer HS, et al. (2008) Antibodies against fetal brain in sera of mothers with autistic

children.

J Neuroimmunol

194:165

–

172.

7. Zimmerman AW, et al. (2007) Maternal antibrain antibodies in autism.

Brain Behav

Immun

21:351

–

357.

8. Atladóttir HO, et al. (2009) Association of family history of autoimmune diseases and

autism spectrum disorders.

Pediatrics

124:687

–

694.

9. Comi AM, Zimmerman AW, Frye VH, Law PA, Peeden JN (1999) Familial clustering of

autoimmune disorders and evaluation of medical risk factors in autism.

J Child Neurol

14:388

–

394.

10. Chess S (1977) Follow-up report on autism in congenital rubella.

J Autism Child

Schizophr

7:69

–

81.

11. Atladóttir HO, et al. (2010) Maternal infection requiring hospitalization during

pregnancy and autism spectrum disorders.

J Autism Dev Disord

40:1423

–

1430.

12. Goines PE, et al. (2011) Increased midgestational IFN-

, IL-4 and IL-5 in women bearing

a child with autism: A case-control study.

Mol Autism

2:13.

13. Abdallah MW, et al. (2012) Amniotic

fl

uid chemokines and autism spectrum disorders:

An exploratory study utilizing a Danish Historic Birth Cohort.

Brain Behav Immun

26:

170

–

176.

14. Dantzer R, O

’

Connor JC, Freund GG, Johnson RW, Kelley KW (2008) From in-

fl

ammation to sickness and depression: When the immune system subjugates the

brain.

Nat Rev Neurosci

9:46

–

56.

15. Capuron L, Miller AH (2011) Immune system to brain signaling: neuro-

psychopharmacological implications.

Pharmacol Ther

130:226

–

238.

16. Segerstrom SC, Miller GE (2004) Psychological stress and the human immune system:

A meta-analytic study of 30 years of inquiry.

Psychol Bull

130:601

–

630.

17. Amor S, Puentes F, Baker D, van der Valk P (2010) In

fl

ammation in neurodegenerative

diseases.

Immunology

129:154

–

169.

18. Shie FS, Chen YH, Chen CH, Ho IK (2011) Neuroimmune pharmacology of neurode-

generative and mental diseases.

J Neuroimmune Pharmacol

6:28

–

40.

19. Patterson PH (2007) Neuroscience. Maternal effects on schizophrenia risk.

Science

318:576

–

577.

20. Dantzer R, O

’

Connor JC, Lawson MA, Kelley KW (2011) In

fl

ammation-associated

depression: From serotonin to kynurenine.

Psychoneuroendocrinology

36:426

–

436.

21. Shi L, et al. (2009) Activation of the maternal immune system alters cerebellar

development in the offspring.

Brain Behav Immun

23:116

–

123.

22. Malkova NV, Yu CZ, Hsiao EY, Moore MJ, Patterson PH (2012) Maternal immune

activation yields offspring displaying mouse versions of the three core symptoms of

autism.

Brain Behav Immun

26:607

–

616.

23. Adkins B, Leclerc C, Marshall-Clarke S (2004) Neonatal adaptive immunity comes of

age.

Nat Rev Immunol

4:553

–

564.

24. Shi C, et al. (2011) Ly6G+ neutrophils are dispensable for defense against systemic

Listeria monocytogenes

infection.

J Immunol

187:5293

–

5298.

25. Perry W, Minassian A, Lopez B, Maron L, Lincoln A (2007) Sensorimotor gating de

fi

cits

in adults with autism.

Biol Psychiatry

61:482

–

486.

26. Ashwood P, et al. (2011) Altered T cell responses in children with autism.

Brain Behav

Immun

25:840

–

849.

27. Mostafa GA, Al Shehab A, Fouad NR (2010) Fre

quency of CD4+CD25high regulatory T cells

in the peripheral blood of Egyptian children with autism.

J Child Neurol

25:328

–

335.

28. Enstrom AM, Van de Water JA, Ashwood P (2009) Autoimmunity in autism.

Curr Opin

Investig Drugs

10:463

–

473.

29. Warren RP, Yonk J, Burger RW, Odell D, Warren WL (1995) DR-positive T cells in

autism: Association with decreased plasma levels of the complement C4B protein.

Neuropsychobiology

31:53

–

57.

30. Plioplys AV, Greaves A, Kazemi K, Silverman E (1994) Lymphocyte function in autism

and Rett syndrome.

Neuropsychobiology

29:12

–

16.

31. Ashwood P, Wake

fi

eld AJ (2006) Immune activation of peripheral blood and mucosal

CD3+ lymphocyte cytokine pro

fi

les in children with autism and gastrointestinal

symptoms.

J Neuroimmunol

173:126

–

134.

32. Ricevuti G, Mazzone A, Pasotti D, de Servi S, Specchia G (1991) Role of granulocytes in

endothelial injury in coronary heart disease in humans.

Atherosclerosis

91:1

–

14.

33. Hansen NE, Karle H, Andersen V, Malmquist J, Hoff GE (1976) Neutrophilic gran-

ulocytes in acute bacterial infection. Sequential studies on lysozyme, myeloperox-

idase and lactoferrin.

Clin Exp Immunol

26:463

–

468.

34. Passegué E, Jochum W, Schorpp-Kistner M, Möhle-Steinlein U, Wagner EF (2001)

Chronic myeloid leukemia with increased granulocyte progenitors in mice lacking

junB expression in the myeloid lineage.

Cell

104:21

–

32.

35. Courtney PA, et al. (1999) Increased apoptotic peripheral blood neutrophils in sys-

temic lupus erythematosus: Relations with disease activity, antibodies to double

stranded DNA, and neutropenia.

Ann Rheum Dis

58:309

–

314.

36. Cascão R, Rosário HS, Souto-Carneiro MM, Fonseca JE (2010) Neutrophils in rheu-

matoid arthritis: More than simple

fi

nal effectors.

Autoimmun Rev

9:531

–

535.

37. Sweeten TL, Posey DJ, McDougle CJ (2003) High blood monocyte counts and neo-

pterin levels in children with autistic disorder.

Am J Psychiatry

160:1691

–

1693.

38. Hsiao EY, Patterson PH (2011) Activation of the maternal immune system induces

endocrine changes in the placenta via IL-6.

Brain Behav Immun

25:604

–

615.

39. Robb L (2007) Cytokine receptors and hematopoietic differentiation.

Oncogene

26:

6715

–

6723.

40. Garay PA, Hsiao EY, Patterson PH, McAllister AK (2012) Maternal immune activation

causes age- and region-speci

fi

c changes in brain cytokines in offspring throughout

development.

Brain Behav Immun

, in press.

41. Chen SK, et al. (2010) Hematopoietic origin of pathological grooming in Hoxb8

mutant mice.

Cell

141:775

–

785.

42. Kwan W, et al. (2012) Bone marrow transplantation confers modest bene

fi

ts in mouse

models of Huntington

’

s disease.

J Neurosci

32:133

–

142.

43. Derecki NC, et al. (2012) Wild-type microglia arrest pathology in a mouse model of

Rett syndrome.

Nature

484:105

–

109.

44. Brynskikh A, Warren T, Zhu J, Kipnis J (2008) Adaptive immunity affects learning

behavior in mice.

Brain Behav Immun

22:861

–

869.

45. Cushman J, Lo J, Huang Z, Wasserfall C, Petitto JM (2003) Neurobehavioral changes

resulting from recombinase activation gene 1 deletion.

Clin Diagn Lab Immunol

10:

–

18.

46. Kipnis J, Cohen H, Cardon M, Ziv Y, Schwartz M (2004) T cell de

fi

ciency leads to

cognitive dysfunction: Implications for therapeutic vaccination for schizophrenia and

other psychiatric conditions.

Proc Natl Acad Sci USA

101:8180

–

8185.

47. Mombaerts P, et al. (1994) Peripheral lymphoid development and function in TCR

mutant mice.

Int Immunol

6:1061

–

1070.

48. Heijtz RD, et al. (2011) Normal gut microbiota modulates brain development and

behavior.

Proc Natl Acad Sci USA

108:3047

–

3052.

49. Heo Y, Zhang Y, Gao D, Miller VM, Lawrence DA (2011) Aberrant immune responses

in a mouse with behavioral disorders.

PLoS ONE

6:e20912.

50. Schneider T, et al. (2008) Gender-speci

fi

c behavioral and immunological alterations

in an animal model of autism induced by prenatal exposure to valproic acid.

Psychoneuroendocrinology

33:728

–

740.

51. Campbell DB, Li C, Sutcliffe JS, Persico AM, Levitt P (2008) Genetic evidence

implicating multiple genes in the MET receptor tyrosine kinase pathway in autism

spectrum disorder.

Autism Res

1:159

–

168.

52. Ashwood P, et al. (2011) Elevated plasma cytokines in autism spectrum disorders

provide evidence of immune dysfunction and are associated with impaired behavioral

outcome.

Brain Behav Immun

25:40

–

45.

53. Heuer L, et al. (2008) Reduced levels of immunoglobulin in children with autism

correlates with behavioral symptoms.

Autism Res

1:275

–

283.

54. Smith SE, Li J, Garbett K, Mirnics K, Patterson PH (2007) Maternal immune activation

alters fetal brain development through interleukin-6.

J Neurosci

27:10695

–

10702.

55. Walf AA, Frye CA (2007) The use of the elevated plus maze as an assay of anxiety-

related behavior in rodents.

Nat Protoc

2:322

–

328.

56. Plappert CF, Kuhn S, Schnitzler HU, Pilz PK (2006) Experience increases the prepulse

inhibition of the acoustic startle response in mice.

Behav Neurosci

120:16

–

23.

Hsiao et al.

PNAS

July 31, 2012

vol. 109

no. 31

12781

NEUROSCIENCE