**1. Introduction**

There is consensus among investigators studying Autism Spectrum Disorders (ASD) that the etiological basis involves environmental factors acting on the genetic susceptibility of the individual [1-5]. Over 100 candidate genes that may contribute to ASD susceptibility have been identified, and numerous environmental "triggers" have been suggested. Yet, the cause of ASD eludes clear definition and most likely is, as in most diseases, multi-factorial. However, several common immunological themes emerge from clinical and experimental studies of ASD, including persistent neuroinflammation, immune dysregulation, or autoim‐ mune manifestations in many autistic children. Thus, in addition to genetic and environ‐ mental factors, there is compelling evidence that immune factors also play a role in ASD. Abnormalities consistent with immune dysregulation, including abnormal or skewed T helper (Th) cell subsets and cytokine profiles, decreased lymphocyte numbers, decreased T cell mitogen responses, and an imbalance of serum immunoglobulin levels have been re‐ ported in children with ASD [6-11].

Recent results of transcriptomic analysis of autistic brains [5] provides strong evidence sup‐ porting a gene-environment etiology for ASD. These authors demonstrated consistent differ‐ ences in transcriptome organization in the cerebral cortex of autistic and normal brains, and identified two discrete modules of co-expressed genes associated with autism. The first, a neuronal module of 209 genes, was enriched for known autism susceptibility genes, and the second module of 235 genes was enriched for immune genes and glial markers. Gene enrich‐ ment analysis showed that genes in the neuronal module were downregulated and enriched

© 2013 Ponzio et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

for gene ontology categories related to synaptic function, whereas the genes in the immuneglial module were upregulated, and showed enrichment for gene ontology categories impli‐ cated in immune and inflammatory responses. The finding of a genetic association for the neuronal module genes, but a non-genetic association for the immune-glial module genes strengthens the gene-environment etiology for ASD.

more frequently in mothers of these autistic children. Our results show that mothers of au‐ tistic children in this cohort have significant increases in pro-inflammatory cytokine gene polymorphisms, thereby conferring the genetic capability to respond more vigorously to im‐ mune stimulation by producing the types and amounts of cytokines that promote inflamma‐ tory reactions. Thus, results obtained from our investigation of the experimental prenatal mouse model of maternal immune stimulation during pregnancy have already shown bio‐

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

**Factor**

Th1 CD4; Tim-3 STAT1 T-bet IL-12 IFN-γ, IL-2 Th2 CD4; T1/ST2 STAT3 GATA3 IL-4 IL-4,5,10,13

Treg CD4; CD25hi FoxP3 FoxP3 TGF-β IL-10, TGF-β

The hypothesis we are investigating in the prenatal mouse model is that maternal immune stimulation during pregnancy acts as a "first hit" that alters the developing immune system in ways that result in more robust pro-inflammatory immune responses by offspring upon subsequent (i.e. second hit) postnatal immune stimulation. Moreover, such fetal program‐ ming occurs in elements of both the innate and adaptive immune systems. Therefore, our experiments investigate how maternal immune stimulation during pregnancy influences the development and function of myeloid and lymphoid compartments of the immune system beginning at the level of the progenitor cells, and progressing to functional outcomes in neo‐ nates and adult offspring. In the myeloid compartment, we are focusing on the functions of Antigen Presenting Cells (APC), and on those innate immune elements that mediate acute inflammatory responses. With respect to the adaptive immune system, we are focusing on pro-inflammatory T helper (Th) cell subsets (Th1 and Th17) and anti-inflammatory Th cell subsets [T regulatory (Treg) cells and Th2 cells]. To do this, we are using several well-charac‐ terized model systems to document the pro-inflammatory nature of the offspring of poly(I:C)-injected vs. PBS-injected pregnant dams. The results of these studies are forming a solid foundation to investigate how the pro-inflammatory phenotype exhibited by these off‐

spring also contributes to the etiology and neuroinflammation associated with ASD.

Th cell subsets are induced by different cytokines, use different cell signaling pathways, and produce unique cytokine profiles mediated by cytokine-specific transcription factors (Table 1). Th17 and Treg cells are dependent on cytokines for their development, maintenance, and function, and have been implicated in modulating the incidence and/or progression of vari‐ ous inflammatory and autoimmune phenomena, including rheumatoid arthritis (RA) [47], Experimental Autoimmune Encephalomyelitis (EAE) [48-50], Inflammatory Bowel Disease

**Inducing Cytokines Cytokines**

http://dx.doi.org/10.5772/53990

115

IL-1, IL-6 TNF-α TGF-β IL-1,6,17,21,22

**produced**

logical relevance in humans.

**Table 1.** Properties of T helper (Th) cell subsets

**Th cell Type Surface Markers Signal Pathways Transcription**

Th17 Not yet defined STAT3 RORγt

Compelling clinical data demonstrate that children of mothers exposed to certain infectious organisms during pregnancy have significantly higher frequencies of neurological disorders [12-21], including schizophrenia and ASD, the etiology of which have been linked to activa‐ tion of the maternal inflammatory/immune responses (reviewed in [9, 22]). Rodent studies in which the maternal immune system is activated during pregnancy replicate these clinical findings, and provide validated mouse models of ASD [14, 15, 19, 23-33]. We have used a well-characterized prenatal mouse model to investigate questions related to the influence of maternal immune stimulation during pregnancy as an environmental risk factor that affects development of the brain and immune system in the offspring.

Injection of pregnant dams with polyclonal immune stimuli, [e.g., polyinosinic-polycytidylic acid (poly(I:C), lipopolysaccharide (LPS)] or direct injection of the pro-inflammatory cyto‐ kines these polyclonal stimuli induce (e.g., IL-1, IL-2, IL-6) cause immune dysregulation and behavioral abnormalities in their offspring in comparison to the offspring of pregnant dams given a control [i.e., Phosphate Buffered Saline (PBS)] injection [30, 34-39]. The underlying mechanisms that mediate these abnormalities have not been clearly defined, and are the fo‐ cus of ongoing studies by us and others. A unique and powerful advantage of this model is the ability to examine subjects for the initiation and persistence of effects and mechanisms over a continuum of time and development from the earliest embryonic stages through the neonatal period and into adulthood.

While it is impossible for any animal model to completely replicate a human condition as complex as ASD, the mouse model of maternal immune stimulation with poly(I:C) has been recognized as an excellent prenatal model for numerous reasons presented in recent reviews by Meyer and Feldon [40] and Patterson [41]. These include (i) **face validity** (resemblance to the human symptoms) (ii) **construct validity** (similarity to the underlying causes of the dis‐ ease) and (iii) **predictive validity** (expected responses to treatments that are effective in the human disease) [42]. Thus, offspring from poly(I:C)-injected dams exhibit behavioral anomalies reminiscent of those seen in autistic and schizophrenic individuals. In addition to their behavioral abnormalities, our studies show that as a result of in utero exposure to products of maternal immune stimulation these offspring also exhibit a "pro-inflammatory" phenotype that confers a vulnerability to develop immune-mediated pathology after birth and into adulthood [43-45].

In this regard, the results obtained from our investigation of the poly(I:C) mouse model have provided the scientific rationale for an ongoing translational research project to deter‐ mine if similar molecular pathogenic mechanisms are involved in a cohort of ASD children who also exhibit diagnostic evidence of immune dysregulation [46]. Using DNA obtained from the Autism Genetic Resource Exchange (AGRE) database, we initiated a parallel study to determine if there were polymorphisms in selected maternal cytokine genes that occurred more frequently in mothers of these autistic children. Our results show that mothers of au‐ tistic children in this cohort have significant increases in pro-inflammatory cytokine gene polymorphisms, thereby conferring the genetic capability to respond more vigorously to im‐ mune stimulation by producing the types and amounts of cytokines that promote inflamma‐ tory reactions. Thus, results obtained from our investigation of the experimental prenatal mouse model of maternal immune stimulation during pregnancy have already shown bio‐ logical relevance in humans.


**Table 1.** Properties of T helper (Th) cell subsets

for gene ontology categories related to synaptic function, whereas the genes in the immuneglial module were upregulated, and showed enrichment for gene ontology categories impli‐ cated in immune and inflammatory responses. The finding of a genetic association for the neuronal module genes, but a non-genetic association for the immune-glial module genes

Compelling clinical data demonstrate that children of mothers exposed to certain infectious organisms during pregnancy have significantly higher frequencies of neurological disorders [12-21], including schizophrenia and ASD, the etiology of which have been linked to activa‐ tion of the maternal inflammatory/immune responses (reviewed in [9, 22]). Rodent studies in which the maternal immune system is activated during pregnancy replicate these clinical findings, and provide validated mouse models of ASD [14, 15, 19, 23-33]. We have used a well-characterized prenatal mouse model to investigate questions related to the influence of maternal immune stimulation during pregnancy as an environmental risk factor that affects

Injection of pregnant dams with polyclonal immune stimuli, [e.g., polyinosinic-polycytidylic acid (poly(I:C), lipopolysaccharide (LPS)] or direct injection of the pro-inflammatory cyto‐ kines these polyclonal stimuli induce (e.g., IL-1, IL-2, IL-6) cause immune dysregulation and behavioral abnormalities in their offspring in comparison to the offspring of pregnant dams given a control [i.e., Phosphate Buffered Saline (PBS)] injection [30, 34-39]. The underlying mechanisms that mediate these abnormalities have not been clearly defined, and are the fo‐ cus of ongoing studies by us and others. A unique and powerful advantage of this model is the ability to examine subjects for the initiation and persistence of effects and mechanisms over a continuum of time and development from the earliest embryonic stages through the

While it is impossible for any animal model to completely replicate a human condition as complex as ASD, the mouse model of maternal immune stimulation with poly(I:C) has been recognized as an excellent prenatal model for numerous reasons presented in recent reviews by Meyer and Feldon [40] and Patterson [41]. These include (i) **face validity** (resemblance to the human symptoms) (ii) **construct validity** (similarity to the underlying causes of the dis‐ ease) and (iii) **predictive validity** (expected responses to treatments that are effective in the human disease) [42]. Thus, offspring from poly(I:C)-injected dams exhibit behavioral anomalies reminiscent of those seen in autistic and schizophrenic individuals. In addition to their behavioral abnormalities, our studies show that as a result of in utero exposure to products of maternal immune stimulation these offspring also exhibit a "pro-inflammatory" phenotype that confers a vulnerability to develop immune-mediated pathology after birth

In this regard, the results obtained from our investigation of the poly(I:C) mouse model have provided the scientific rationale for an ongoing translational research project to deter‐ mine if similar molecular pathogenic mechanisms are involved in a cohort of ASD children who also exhibit diagnostic evidence of immune dysregulation [46]. Using DNA obtained from the Autism Genetic Resource Exchange (AGRE) database, we initiated a parallel study to determine if there were polymorphisms in selected maternal cytokine genes that occurred

strengthens the gene-environment etiology for ASD.

114 Recent Advances in Autism Spectrum Disorders - Volume I

development of the brain and immune system in the offspring.

neonatal period and into adulthood.

and into adulthood [43-45].

The hypothesis we are investigating in the prenatal mouse model is that maternal immune stimulation during pregnancy acts as a "first hit" that alters the developing immune system in ways that result in more robust pro-inflammatory immune responses by offspring upon subsequent (i.e. second hit) postnatal immune stimulation. Moreover, such fetal program‐ ming occurs in elements of both the innate and adaptive immune systems. Therefore, our experiments investigate how maternal immune stimulation during pregnancy influences the development and function of myeloid and lymphoid compartments of the immune system beginning at the level of the progenitor cells, and progressing to functional outcomes in neo‐ nates and adult offspring. In the myeloid compartment, we are focusing on the functions of Antigen Presenting Cells (APC), and on those innate immune elements that mediate acute inflammatory responses. With respect to the adaptive immune system, we are focusing on pro-inflammatory T helper (Th) cell subsets (Th1 and Th17) and anti-inflammatory Th cell subsets [T regulatory (Treg) cells and Th2 cells]. To do this, we are using several well-charac‐ terized model systems to document the pro-inflammatory nature of the offspring of poly(I:C)-injected vs. PBS-injected pregnant dams. The results of these studies are forming a solid foundation to investigate how the pro-inflammatory phenotype exhibited by these off‐ spring also contributes to the etiology and neuroinflammation associated with ASD.

Th cell subsets are induced by different cytokines, use different cell signaling pathways, and produce unique cytokine profiles mediated by cytokine-specific transcription factors (Table 1). Th17 and Treg cells are dependent on cytokines for their development, maintenance, and function, and have been implicated in modulating the incidence and/or progression of vari‐ ous inflammatory and autoimmune phenomena, including rheumatoid arthritis (RA) [47], Experimental Autoimmune Encephalomyelitis (EAE) [48-50], Inflammatory Bowel Disease (IBD) [51-53], diabetes [54, 55], and atherosclerosis [56, 57]. Thus far, however, little is known about the involvement of proinflammatory Th1 and Th17 cells in autism, and how Th cell subsets interact with microglial APC in the brain [58].

jected dams exhibit a pro-inflammatory phenotype in comparison to offspring of PBSinjected dams. In addition, however, T helper (Th) cells from offspring of immune poly(I:C) injected dams show a unique ability to preferentially differentiate to become pro-

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

http://dx.doi.org/10.5772/53990

117

Figures 2A and 2B show the significant increases in IL-6 at 2hr and 16hr after poly(I:C) injec‐ tion, and similar differences were also seen in levels of IL-1β, IL-12, TNF-α, and GM-CSF in

**Figure 2. IL-6 levels in sera and amniotic fluids from B6 pregnant dams.** Samples collected 2hrs after injection of poly(I:C) or PBS were tested for IL-6 in sera (A) and amniotic fluids (B) using Luminex bead-based multiplex assay. Data

In addition to testing sera and amniotic fluids from pregnant dams, we also analyze pheno‐ typic and functional characteristics of lymphoid cells from offspring. To avoid bias due to "litter effects" [61], the number of subjects examined in our experiments not only reflects off‐ spring within a litter, but also offspring from multiple dams, so that the "N" in our studies

considers both the number of dams, as well as the number of offspring.

show mean ± SEM. (N=3-8; \*\*p<0.01 using Tukey's HSD test )

inflammatory Th17 cells.

these samples [45, 60].


**Figure 1.** Prenatal models of maternal immune stimulation during pregnancy

#### **2. Prenatal models of immune stimulation using poly(I:C)**

In the prenatal model of maternal immune stimulation with poly(I:C) (Figure 1), C57BL/6 (B6) females and males are mated, and appearance of a vaginal plug is considered day zero of gestation (E0). At E12, pregnant females are given a single i.p. injection of poly(I:C) or PBS alone as a control. Sera and amniotic fluids are harvested, and stored at -80o C prior to measurement of cytokine levels by Luminex® bead-based multiplex assay [59] that meas‐ ures up to 32 individual cytokines. In vitro and in vivo analyses are also performed on the neonatal and adult offspring of these poly(I:C)-injected and PBS-injected pregnant dams to assess the phenotype and function of their innate and adaptive immune system compo‐ nents. As also shown in Figure 1, we mate females that are immunologically naïve (i.e., nonimmune), as well as females that possess immunological memory (i.e., immune) with immunologically naïve males. The phenotype of the offspring from these mating schemes reflects the immune status of the of the pregnant dams, and the nature of the prenatal stimu‐ lus. Our results demonstrate that offspring of both non-immune and immune poly(I:C)-in‐ jected dams exhibit a pro-inflammatory phenotype in comparison to offspring of PBSinjected dams. In addition, however, T helper (Th) cells from offspring of immune poly(I:C) injected dams show a unique ability to preferentially differentiate to become proinflammatory Th17 cells.

(IBD) [51-53], diabetes [54, 55], and atherosclerosis [56, 57]. Thus far, however, little is known about the involvement of proinflammatory Th1 and Th17 cells in autism, and how

Th cell subsets interact with microglial APC in the brain [58].

116 Recent Advances in Autism Spectrum Disorders - Volume I

**Figure 1.** Prenatal models of maternal immune stimulation during pregnancy

**2. Prenatal models of immune stimulation using poly(I:C)**

In the prenatal model of maternal immune stimulation with poly(I:C) (Figure 1), C57BL/6 (B6) females and males are mated, and appearance of a vaginal plug is considered day zero of gestation (E0). At E12, pregnant females are given a single i.p. injection of poly(I:C) or

measurement of cytokine levels by Luminex® bead-based multiplex assay [59] that meas‐ ures up to 32 individual cytokines. In vitro and in vivo analyses are also performed on the neonatal and adult offspring of these poly(I:C)-injected and PBS-injected pregnant dams to assess the phenotype and function of their innate and adaptive immune system compo‐ nents. As also shown in Figure 1, we mate females that are immunologically naïve (i.e., nonimmune), as well as females that possess immunological memory (i.e., immune) with immunologically naïve males. The phenotype of the offspring from these mating schemes reflects the immune status of the of the pregnant dams, and the nature of the prenatal stimu‐ lus. Our results demonstrate that offspring of both non-immune and immune poly(I:C)-in‐

C prior to

PBS alone as a control. Sera and amniotic fluids are harvested, and stored at -80o

Figures 2A and 2B show the significant increases in IL-6 at 2hr and 16hr after poly(I:C) injec‐ tion, and similar differences were also seen in levels of IL-1β, IL-12, TNF-α, and GM-CSF in these samples [45, 60].

**Figure 2. IL-6 levels in sera and amniotic fluids from B6 pregnant dams.** Samples collected 2hrs after injection of poly(I:C) or PBS were tested for IL-6 in sera (A) and amniotic fluids (B) using Luminex bead-based multiplex assay. Data show mean ± SEM. (N=3-8; \*\*p<0.01 using Tukey's HSD test )

In addition to testing sera and amniotic fluids from pregnant dams, we also analyze pheno‐ typic and functional characteristics of lymphoid cells from offspring. To avoid bias due to "litter effects" [61], the number of subjects examined in our experiments not only reflects off‐ spring within a litter, but also offspring from multiple dams, so that the "N" in our studies considers both the number of dams, as well as the number of offspring.

*Use of immunologically naïve pregnant dams:* Immunologically naïve mice are used by most in‐ vestigators in the standard prenatal model of immune stimulation during pregnancy. These pregnant dams are injected with poly(I:C) on embryonic day 12 (E12), and control pregnant dams are injected with PBS. The embryos and offspring from these dams are then used ex‐ perimentally to determine the influence of maternal immune stimulation on prenatal devel‐ opment and postnatal function. Since this model was originally developed to investigate neurodevelopmental disorders, such as schizophrenia and autism, a majority of the studies focus on the CNS and behavioral outcomes of offspring. These investigations have shown that maternal immune stimulation during pregnancy with polyclonal stimuli [e.g., poly(I:C) or LPS], infectious pathogens, or specific cytokines (e.g., IL-2 or IL-6) results in expression of ASD-like behavioral manifestations, as well as structural or functional changes in cells in the brain of the offspring [39-41, 61, 62].

However, in the prenatal models that use poly(I:C) as the immune stimulus, the type of poly(I:C) (i.e., sodium or potassium salt), dose of poly(I:C) (2-20 mg/Kg), and time of in‐ jection during pregnancy (E9 through E18) can influence some of the parameters that have been examined in these offspring, including open field exploration, sensorimotor gating (e.g., prepulse inhibition of the startle response), and repetitive/perserverative be‐ havior ([63, 64]. It is thought that poly(I:C)-induced maternal cytokines are primarily re‐ sponsible for the abnormalities seen in offspring. However, downstream effects induced by these maternal cytokines or trans-placental stimulation of fetal tissues by poly(I:C) it‐ self have not been completely ruled out.

*Use of pregnant dams with immunological memory:* In addition to the existing model using immunologically naïve dams, we also modified this mouse model of neurodevelopmental disorders by using dams that possess immunological memory prior to mating [43, 44]. This experimental design more closely resembles the human scenario, where women pos‐ sess immunological memory resulting from immunizations and natural exposure to envi‐ ronmental antigens prior to pregnancy. Using dams with immunological memory yields a more robust mouse prenatal model, which revealed outcomes in offspring that may be significant not only in the etiology and/or pathogenesis of schizophrenia and autism, but also in other disorders that are currently not being considered by use of these prenatal mouse models.

**Figure 3. Enhanced production of Th17 cells in offspring poly(I:C)-injected (20mg/Kg) immune dams.** Spleen cells from 3wk old offspring of poly(I:C)- and PBS-injected dams were stimulated with 3ng/ml PMA and 100ng/ml ion‐ omycin for 16hr, the last 4hr of which were in the presence of 10ug/ml Brefeldin A to block cytokine secretion. Cells were harvested, and stained with fluorochrome-conjugated mAbs to detect cell surface molecules and intracellular cytokines by FACS analysis. The spleen cells analyzed in each of the panels were from offspring of PBS-injected immu‐ nologically naïve dams (A); offspring of poly(I:C)-injected immunologically naïve dams (B); offspring of PBS-injected immune dams (C), and offspring of poly(I:C)-injected immune dams (D). Numbers in upper left quadrants are percen‐ tages of IL-17A+ (Th17) cells after gating on CD4+ cells. Results shown are representative of seven experiments com‐ paring 18 offspring from 12 different dams. Overall results of percentages of Th17 cells were: 15.1±7.8 in offspring from immune poly(I:C)-injected dams vs. 0.8±0.5 in offspring from immune PBS-injected dams (p=0.05 using Tukey's

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

http://dx.doi.org/10.5772/53990

119

The offspring of poly(I:C)-injected (vs. PBS-injected) pregnant dams who possess immuno‐ logical memory prior to pregnancy exhibit a unique pro-inflammatory phenotype in which there is preferential development of Th17 lymphocytes after T cell activation (Figure 3) [43, 44]. This preferential Th17 cell development is not seen at all in offspring of immunological‐ ly naïve poly(I:C)-injected or PBS-injected pregnant dams. Given their role in immune-medi‐ ated disorders, it is likely that the potential to produce Th17 cells that we have discovered in offspring of poly(I:C)-injected pregnant dams with immunological memory may also be an important component in the neuroinflammatory pathogenesis of ASD-like changes that have been observed in this prenatal mouse model. Thus, one hypothesis we have tested is that the pro-inflammatory phenotype of offspring induced as a result of embryonic develop‐ ment in a pro-inflammatory cytokine environment in utero make them more susceptible

HSD test )

In both of these models, we and others have previously shown that following injection of poly(I:C), pregnant dams produce significantly higher levels of pro-inflammatory cyto‐ kines (e.g., IL-1, IL-6, IL-12, TNF-α, and GM-CSF) than PBS-injected dams in sera as well as amniotic fluids. Most of the studies involving structural/chemical changes and behav‐ ioral abnormalities that are observed after injection of poly(I:C) to pregnant dams have been performed on adult offspring from immunologically naïve pregnant dams. Recent‐ ly, Hsaio, et al. [65] observed alterations in the peripheral immune system of these off‐ spring. Our results indicate that the adult offspring of immunologically naïve poly(I:C) injected pregnant dams also exhibit a more robust acute inflammatory response after injection of the TLR2 ligand, zymosan [45, 60].

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy http://dx.doi.org/10.5772/53990 119

*Use of immunologically naïve pregnant dams:* Immunologically naïve mice are used by most in‐ vestigators in the standard prenatal model of immune stimulation during pregnancy. These pregnant dams are injected with poly(I:C) on embryonic day 12 (E12), and control pregnant dams are injected with PBS. The embryos and offspring from these dams are then used ex‐ perimentally to determine the influence of maternal immune stimulation on prenatal devel‐ opment and postnatal function. Since this model was originally developed to investigate neurodevelopmental disorders, such as schizophrenia and autism, a majority of the studies focus on the CNS and behavioral outcomes of offspring. These investigations have shown that maternal immune stimulation during pregnancy with polyclonal stimuli [e.g., poly(I:C) or LPS], infectious pathogens, or specific cytokines (e.g., IL-2 or IL-6) results in expression of ASD-like behavioral manifestations, as well as structural or functional changes in cells in the

However, in the prenatal models that use poly(I:C) as the immune stimulus, the type of poly(I:C) (i.e., sodium or potassium salt), dose of poly(I:C) (2-20 mg/Kg), and time of in‐ jection during pregnancy (E9 through E18) can influence some of the parameters that have been examined in these offspring, including open field exploration, sensorimotor gating (e.g., prepulse inhibition of the startle response), and repetitive/perserverative be‐ havior ([63, 64]. It is thought that poly(I:C)-induced maternal cytokines are primarily re‐ sponsible for the abnormalities seen in offspring. However, downstream effects induced by these maternal cytokines or trans-placental stimulation of fetal tissues by poly(I:C) it‐

*Use of pregnant dams with immunological memory:* In addition to the existing model using immunologically naïve dams, we also modified this mouse model of neurodevelopmental disorders by using dams that possess immunological memory prior to mating [43, 44]. This experimental design more closely resembles the human scenario, where women pos‐ sess immunological memory resulting from immunizations and natural exposure to envi‐ ronmental antigens prior to pregnancy. Using dams with immunological memory yields a more robust mouse prenatal model, which revealed outcomes in offspring that may be significant not only in the etiology and/or pathogenesis of schizophrenia and autism, but also in other disorders that are currently not being considered by use of these prenatal

In both of these models, we and others have previously shown that following injection of poly(I:C), pregnant dams produce significantly higher levels of pro-inflammatory cyto‐ kines (e.g., IL-1, IL-6, IL-12, TNF-α, and GM-CSF) than PBS-injected dams in sera as well as amniotic fluids. Most of the studies involving structural/chemical changes and behav‐ ioral abnormalities that are observed after injection of poly(I:C) to pregnant dams have been performed on adult offspring from immunologically naïve pregnant dams. Recent‐ ly, Hsaio, et al. [65] observed alterations in the peripheral immune system of these off‐ spring. Our results indicate that the adult offspring of immunologically naïve poly(I:C) injected pregnant dams also exhibit a more robust acute inflammatory response after

brain of the offspring [39-41, 61, 62].

118 Recent Advances in Autism Spectrum Disorders - Volume I

self have not been completely ruled out.

injection of the TLR2 ligand, zymosan [45, 60].

mouse models.

**Figure 3. Enhanced production of Th17 cells in offspring poly(I:C)-injected (20mg/Kg) immune dams.** Spleen cells from 3wk old offspring of poly(I:C)- and PBS-injected dams were stimulated with 3ng/ml PMA and 100ng/ml ion‐ omycin for 16hr, the last 4hr of which were in the presence of 10ug/ml Brefeldin A to block cytokine secretion. Cells were harvested, and stained with fluorochrome-conjugated mAbs to detect cell surface molecules and intracellular cytokines by FACS analysis. The spleen cells analyzed in each of the panels were from offspring of PBS-injected immu‐ nologically naïve dams (A); offspring of poly(I:C)-injected immunologically naïve dams (B); offspring of PBS-injected immune dams (C), and offspring of poly(I:C)-injected immune dams (D). Numbers in upper left quadrants are percen‐ tages of IL-17A+ (Th17) cells after gating on CD4+ cells. Results shown are representative of seven experiments com‐ paring 18 offspring from 12 different dams. Overall results of percentages of Th17 cells were: 15.1±7.8 in offspring from immune poly(I:C)-injected dams vs. 0.8±0.5 in offspring from immune PBS-injected dams (p=0.05 using Tukey's HSD test )

The offspring of poly(I:C)-injected (vs. PBS-injected) pregnant dams who possess immuno‐ logical memory prior to pregnancy exhibit a unique pro-inflammatory phenotype in which there is preferential development of Th17 lymphocytes after T cell activation (Figure 3) [43, 44]. This preferential Th17 cell development is not seen at all in offspring of immunological‐ ly naïve poly(I:C)-injected or PBS-injected pregnant dams. Given their role in immune-medi‐ ated disorders, it is likely that the potential to produce Th17 cells that we have discovered in offspring of poly(I:C)-injected pregnant dams with immunological memory may also be an important component in the neuroinflammatory pathogenesis of ASD-like changes that have been observed in this prenatal mouse model. Thus, one hypothesis we have tested is that the pro-inflammatory phenotype of offspring induced as a result of embryonic develop‐ ment in a pro-inflammatory cytokine environment in utero make them more susceptible (i.e., vulnerable) to develop immune-mediated pathology. Indeed, we have obtained com‐ pelling results from in vivo experiments in adult offspring that strongly support this possi‐ bility. Using a model of EAE, in which mice are injected with an encephalogenic-peptide, Myelin Oligodendrocyte Glycoprotein peptide (MOG35-55), we found that adult offspring of poly(I:C)-injected pregnant dams exhibited a significantly higher frequency and earlier on‐ set of clinical symptoms of EAE compared to offspring of PBS-injected pregnant dams [45, 60]. Our zymosan induced results and the EAE experiments are described in subsequent sec‐ tions of this chapter.

*Maternal vs. fetal sources of cytokines:* In this prenatal model, a single i.p. injection of poly(I:C) (or control PBS) is given on gestational day 12 (E12). Convincing evidence from this model by us and others [30, 43, 44, 61, 66-68] has shown that pro-inflammatory cyto‐ kines (IL-1, IL-6, IL-12, TNF-α, GM-CSF) produced as a result of maternal immune stim‐ ulation during pregnancy induce changes in the development of the immune system and the brain of offspring that result in immunological and behavioral manifestations similar to those seen in individuals with ASD. To what degree these changes are induced by cy‐ tokines produced by the mother or fetus has not been fully defined. However, our re‐ sults using IL-6 knock-out (KO) dams mated with wild type males [44], suggests that there is a fetal source for at least some of the cytokines detected in the amniotic fluid of poly(I:C)-injected pregnant dams.

**Figure 4. Source of cytokines in pregnant dams.** Sera (N=6) and amniotic fluids (N=17) from IL-6 KO pregnant dams were collected 24 hrs after injection of poly(I:C), and IL-6 levels determined by Luminex assay. (\*p<0.0001 Tukey's HSD

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

http://dx.doi.org/10.5772/53990

121

**Figure 5. Source of cytokines in offspring.** Spleen cells from WT B6 (N=5) and spleen and placental cells from poly(I:C)-injected IL-6 KO females (N=5) were cultured with/without PMA and Ionomycin (P/I). Supernatants were col‐ lected 24hrs later, and tested for the presence of IL-6 by Luminex assay. (\*p<0.02; \*\*p<0.01; Tukey's HSD test)

Regardless of source, however, since in utero exposure to the products of maternal immune stimulation during pregnancy appear to be part of the underlying mechanisms responsible for the changes observed in offspring, it is important to be sure that the pregnant dam responds to the immune stimulus if their offspring are used for experiments. We have addressed this issue by monitoring locomotor activity in a novel environment in every pregnant dam before, and at 2hrs and 16hrs after injection as a reliable, non-invasive measure of response to poly(I:C). We opted to use this method in lieu of more invasive procedures that would jeopardize pregnancy in these dams and/or add a level of stress that could influence the cytokine levels and/or fetal development. As shown in Figure 6 and Table 2, there is a consistent and dramatic decrease in activity (indicative of "sickness behavior – [71-74]) in poly(I:C)-injected pregnant dams at 2hrs post injection that is not seen in PBS-injected dams. Moreover, sickness behavior at 2hrs post poly(I:C) injection correlates very nicely with the increased levels of pro-inflammatory cyto‐ kines seen at 2hrs in the sera and amniotic fluids of pregnant dams (Figure 2). Activity scores

test).

In these experiments, our results from mating IL-6 knock-out (KO) B6 females (IL-6-/-) and wild-type (WT) B6 males (IL-6+/+) show that despite a maternal genetic deficiency for IL-6 production, fetal components of the heterozygous IL-6+/- placenta are a source of this cytokine (Figures 4 and 5), and heterozygous neonates can also produce IL-6 [44]. Using similar mating schemes in this prenatal model, however, Hsiao, et al. [66] did not find IL-6 in amniotic fluid of poly(I:C)-injected pregnant IL-6 KO B6 dams. Our results sug‐ gest that poly(I:C) (a TLR3 agonist) stimulates fetal placental tissues directly, and contrib‐ utes to the levels of IL-6 found in amniotic fluid. This is relevant to the interpretation of data about the source of IL-6 (as well as other cytokines) found in the amniotic fluids and fetal tissues, such as the brain [9, 63, 69], and also because TLR3 is expressed in the brain during fetal development [70].


Pregnant dams were analyzed for sickness behavior before and 2 and 24 hrs after poly(I:C) injection. All mice in a group were analyzed by calculating a ratio, where each post injection sickness behavior score was divided by its preinjection score. The individual ratios were then used to calculate the means, standard errors, and significance values. \*p < 0.0001 (Tukey's HSD test).

**Table 2.** Sickness behavior scores of immune poly(I:C)- and PBS- injected pregnant dams

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy http://dx.doi.org/10.5772/53990 121

(i.e., vulnerable) to develop immune-mediated pathology. Indeed, we have obtained com‐ pelling results from in vivo experiments in adult offspring that strongly support this possi‐ bility. Using a model of EAE, in which mice are injected with an encephalogenic-peptide, Myelin Oligodendrocyte Glycoprotein peptide (MOG35-55), we found that adult offspring of poly(I:C)-injected pregnant dams exhibited a significantly higher frequency and earlier on‐ set of clinical symptoms of EAE compared to offspring of PBS-injected pregnant dams [45, 60]. Our zymosan induced results and the EAE experiments are described in subsequent sec‐

*Maternal vs. fetal sources of cytokines:* In this prenatal model, a single i.p. injection of poly(I:C) (or control PBS) is given on gestational day 12 (E12). Convincing evidence from this model by us and others [30, 43, 44, 61, 66-68] has shown that pro-inflammatory cyto‐ kines (IL-1, IL-6, IL-12, TNF-α, GM-CSF) produced as a result of maternal immune stim‐ ulation during pregnancy induce changes in the development of the immune system and the brain of offspring that result in immunological and behavioral manifestations similar to those seen in individuals with ASD. To what degree these changes are induced by cy‐ tokines produced by the mother or fetus has not been fully defined. However, our re‐ sults using IL-6 knock-out (KO) dams mated with wild type males [44], suggests that there is a fetal source for at least some of the cytokines detected in the amniotic fluid of

In these experiments, our results from mating IL-6 knock-out (KO) B6 females (IL-6-/-) and wild-type (WT) B6 males (IL-6+/+) show that despite a maternal genetic deficiency for IL-6 production, fetal components of the heterozygous IL-6+/- placenta are a source of this cytokine (Figures 4 and 5), and heterozygous neonates can also produce IL-6 [44]. Using similar mating schemes in this prenatal model, however, Hsiao, et al. [66] did not find IL-6 in amniotic fluid of poly(I:C)-injected pregnant IL-6 KO B6 dams. Our results sug‐ gest that poly(I:C) (a TLR3 agonist) stimulates fetal placental tissues directly, and contrib‐ utes to the levels of IL-6 found in amniotic fluid. This is relevant to the interpretation of data about the source of IL-6 (as well as other cytokines) found in the amniotic fluids and fetal tissues, such as the brain [9, 63, 69], and also because TLR3 is expressed in the

**Pregnant dams injected with Sickness behavior ratio N**

Pregnant dams were analyzed for sickness behavior before and 2 and 24 hrs after poly(I:C) injection. All mice in a group were analyzed by calculating a ratio, where each post injection sickness behavior score was divided by its preinjection score. The individual ratios were then used to calculate the means, standard errors, and significance values.

**Table 2.** Sickness behavior scores of immune poly(I:C)- and PBS- injected pregnant dams

PBS 1.00 ± 0.10 9

Poly(I:C) 0.40± 0.02\* 21

tions of this chapter.

120 Recent Advances in Autism Spectrum Disorders - Volume I

poly(I:C)-injected pregnant dams.

brain during fetal development [70].

\*p < 0.0001 (Tukey's HSD test).

**Figure 4. Source of cytokines in pregnant dams.** Sera (N=6) and amniotic fluids (N=17) from IL-6 KO pregnant dams were collected 24 hrs after injection of poly(I:C), and IL-6 levels determined by Luminex assay. (\*p<0.0001 Tukey's HSD test).

**Figure 5. Source of cytokines in offspring.** Spleen cells from WT B6 (N=5) and spleen and placental cells from poly(I:C)-injected IL-6 KO females (N=5) were cultured with/without PMA and Ionomycin (P/I). Supernatants were col‐ lected 24hrs later, and tested for the presence of IL-6 by Luminex assay. (\*p<0.02; \*\*p<0.01; Tukey's HSD test)

Regardless of source, however, since in utero exposure to the products of maternal immune stimulation during pregnancy appear to be part of the underlying mechanisms responsible for the changes observed in offspring, it is important to be sure that the pregnant dam responds to the immune stimulus if their offspring are used for experiments. We have addressed this issue by monitoring locomotor activity in a novel environment in every pregnant dam before, and at 2hrs and 16hrs after injection as a reliable, non-invasive measure of response to poly(I:C). We opted to use this method in lieu of more invasive procedures that would jeopardize pregnancy in these dams and/or add a level of stress that could influence the cytokine levels and/or fetal development. As shown in Figure 6 and Table 2, there is a consistent and dramatic decrease in activity (indicative of "sickness behavior – [71-74]) in poly(I:C)-injected pregnant dams at 2hrs post injection that is not seen in PBS-injected dams. Moreover, sickness behavior at 2hrs post poly(I:C) injection correlates very nicely with the increased levels of pro-inflammatory cyto‐ kines seen at 2hrs in the sera and amniotic fluids of pregnant dams (Figure 2). Activity scores are measured in every pregnant dam, including those that are brought to term and give birth. In this way, we are confident that the offspring used for subsequent in vivo and in vitro experi‐ ments to characterize phenotypic and functional immunological parameters were exposed in utero to a pro-inflammatory cytokine milieu.

TLRs in the developing embryos and offspring is an important question that allows the de‐

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

http://dx.doi.org/10.5772/53990

123

Modulation and desensitization of TLR expression, as well as cross-talk among TLRs has been shown in cells from humans and rodents. There is increasing evidence of TLR expres‐ sion during embryonic development in the placenta, fetal brain and hematopoietic stem cells [78-81]. In this regard, we have obtained results indicating modulation of TLR expres‐ sion in 4wk old neonates from poly(I:C)-injected dams (Figure 7). Using pathway-focused gene expression profiling qRT-PCR arrays, spleen cells from offspring of pregnant dams in‐ jected with the TLR3 agonist, poly(I:C), showed a 3.3 to 4.7 -fold increase in constitutive ex‐ pression levels of TLR2, 4 and 7 over those seen in age-matched control B6 offspring. In contrast, expression levels of TLR3 and 9 were <2-fold greater than controls. These results indicate that exposure to poly(I:C) (or poly(I:C)-induced cytokines) during fetal develop‐ ment results in altered TLR expression that persists after birth, consequences of which may relate to differential immune responses to micro-organisms and auto-antigens. These data also suggest that TLR modulation, desensitization, and cross-talk also occur when fetal tis‐

**Figure 7. Altered constitutive expression of TLRs in offspring of poly(I:C)-injected dams.** RNA was extracted di‐ rectly from unstimulated spleen cells of 4wk old offspring of poly(I:C)- and PBS-injected dams, and tested for expres‐ sion of TLRs using the RT2 Profiler PCR Array system. Data are expressed as fold-changes in gene expression for TLRs in offspring of poly(I:C)-injected dams over PBS-injected dams. The results show >3-fold upregulation for TLR2, 4, and 7

Recent studies have shown expression of TLRs by neural progenitor cells (NPC), neurons and glial cells in the adult brain, which may be important in the responses of these cells to injury or infection [76, 82-88]. Studies of TLR expression in the developing rodent brain have revealed that TLR3 appears as early as embryonic day 12.5 (E12.5) in mouse cortices, but de‐ clines over time. By contrast, TLR2 expression appears around E15.5 and increases with time [70, 88]. Moreover, in standard in vitro neurosphere assays (used to assess developmental potential of NPCs), both TLR2 and TLR3 activation appear to regulate NPC proliferation. These studies raise questions regarding the expression of other TLRs during brain develop‐

and <2-fold increase for TLR3 and 9 in offspring of poly(I:C)- compared to PBS-injected dams.

sign of experiments that address underlying mechanisms.

sues are exposed to TLR agonists in utero.

**Figure 6. Sickness behavior scores in poly(I:C)-injected pregnant dams.** Dams were tested before, and at 2hrs & 16hrs after poly(I:C) or PBS injection. Activity was monitored in a novel environment, and mice were given positive scores for locomotion, rearing, grooming, sniffing, and negative scores for periods of inactivity. The data in this figure show scores for 6 individual poly(I:C)-injected dams at the 3 time periods.

*Effects of maternal immune stimulation on Toll-Like Receptors (TLR):* We are also examining the effects of poly(I:C) exposure during pregnancy on the expression and function of TLRs dur‐ ing fetal development and in neonates and adult offspring. TLRs are part of a larger family of membrane bound cell surface and intracellular Pattern Recognition Receptors (PRR). Eleven TLRs have been discovered in humans (TLRs 1-11), and 12 TLRs have been found in mice (TLRs 1-12). First discovered in Drosophila [75], this family of molecules is very hetero‐ genous, complex, and highly conserved among species. Individual TLRs bind to particular microbial products, such as LPS, peptidoglycan, lipoproteins, and flagellin on bacteria, as well as viral fusion protein, unmethylated CpG motifs, double- and single-stranded RNAs [76, 77]. TLR expression by cells of the innate and adaptive immune systems allows these cells to recognize and respond to extracellular and intracellular microbial pathogens. Down‐ stream cell signaling pathways are initiated when ligands bind to TLRs, leading to activa‐ tion of different transcription factors (e.g., NF-κB and others), which stimulate expression of pro-inflammatory cytokine genes (e.g., IL-1, IL-6, TNF, interferons).

Thus, TLRs play an early and important role at the interface between the environment and host tissues by initiating immune responses against pathogens. In addition to expression on cells of the innate and adaptive immune systems, TLRs are also expressed in/on many cell types in various other tissues of the body, including the placenta, embryonic brain, and hematopoietic progenitor cells. In the context of our experimental model of maternal im‐ mune stimulation during pregnancy, how the maternal response to poly(I:C) (a TLR3 ago‐ nist) during pregnancy affects the normal expression and function of TLR3, as well as other TLRs in the developing embryos and offspring is an important question that allows the de‐ sign of experiments that address underlying mechanisms.

are measured in every pregnant dam, including those that are brought to term and give birth. In this way, we are confident that the offspring used for subsequent in vivo and in vitro experi‐ ments to characterize phenotypic and functional immunological parameters were exposed in

**Figure 6. Sickness behavior scores in poly(I:C)-injected pregnant dams.** Dams were tested before, and at 2hrs & 16hrs after poly(I:C) or PBS injection. Activity was monitored in a novel environment, and mice were given positive scores for locomotion, rearing, grooming, sniffing, and negative scores for periods of inactivity. The data in this figure

*Effects of maternal immune stimulation on Toll-Like Receptors (TLR):* We are also examining the effects of poly(I:C) exposure during pregnancy on the expression and function of TLRs dur‐ ing fetal development and in neonates and adult offspring. TLRs are part of a larger family of membrane bound cell surface and intracellular Pattern Recognition Receptors (PRR). Eleven TLRs have been discovered in humans (TLRs 1-11), and 12 TLRs have been found in mice (TLRs 1-12). First discovered in Drosophila [75], this family of molecules is very hetero‐ genous, complex, and highly conserved among species. Individual TLRs bind to particular microbial products, such as LPS, peptidoglycan, lipoproteins, and flagellin on bacteria, as well as viral fusion protein, unmethylated CpG motifs, double- and single-stranded RNAs [76, 77]. TLR expression by cells of the innate and adaptive immune systems allows these cells to recognize and respond to extracellular and intracellular microbial pathogens. Down‐ stream cell signaling pathways are initiated when ligands bind to TLRs, leading to activa‐ tion of different transcription factors (e.g., NF-κB and others), which stimulate expression of

Thus, TLRs play an early and important role at the interface between the environment and host tissues by initiating immune responses against pathogens. In addition to expression on cells of the innate and adaptive immune systems, TLRs are also expressed in/on many cell types in various other tissues of the body, including the placenta, embryonic brain, and hematopoietic progenitor cells. In the context of our experimental model of maternal im‐ mune stimulation during pregnancy, how the maternal response to poly(I:C) (a TLR3 ago‐ nist) during pregnancy affects the normal expression and function of TLR3, as well as other

utero to a pro-inflammatory cytokine milieu.

122 Recent Advances in Autism Spectrum Disorders - Volume I

show scores for 6 individual poly(I:C)-injected dams at the 3 time periods.

pro-inflammatory cytokine genes (e.g., IL-1, IL-6, TNF, interferons).

Modulation and desensitization of TLR expression, as well as cross-talk among TLRs has been shown in cells from humans and rodents. There is increasing evidence of TLR expres‐ sion during embryonic development in the placenta, fetal brain and hematopoietic stem cells [78-81]. In this regard, we have obtained results indicating modulation of TLR expres‐ sion in 4wk old neonates from poly(I:C)-injected dams (Figure 7). Using pathway-focused gene expression profiling qRT-PCR arrays, spleen cells from offspring of pregnant dams in‐ jected with the TLR3 agonist, poly(I:C), showed a 3.3 to 4.7 -fold increase in constitutive ex‐ pression levels of TLR2, 4 and 7 over those seen in age-matched control B6 offspring. In contrast, expression levels of TLR3 and 9 were <2-fold greater than controls. These results indicate that exposure to poly(I:C) (or poly(I:C)-induced cytokines) during fetal develop‐ ment results in altered TLR expression that persists after birth, consequences of which may relate to differential immune responses to micro-organisms and auto-antigens. These data also suggest that TLR modulation, desensitization, and cross-talk also occur when fetal tis‐ sues are exposed to TLR agonists in utero.

**Figure 7. Altered constitutive expression of TLRs in offspring of poly(I:C)-injected dams.** RNA was extracted di‐ rectly from unstimulated spleen cells of 4wk old offspring of poly(I:C)- and PBS-injected dams, and tested for expres‐ sion of TLRs using the RT2 Profiler PCR Array system. Data are expressed as fold-changes in gene expression for TLRs in offspring of poly(I:C)-injected dams over PBS-injected dams. The results show >3-fold upregulation for TLR2, 4, and 7 and <2-fold increase for TLR3 and 9 in offspring of poly(I:C)- compared to PBS-injected dams.

Recent studies have shown expression of TLRs by neural progenitor cells (NPC), neurons and glial cells in the adult brain, which may be important in the responses of these cells to injury or infection [76, 82-88]. Studies of TLR expression in the developing rodent brain have revealed that TLR3 appears as early as embryonic day 12.5 (E12.5) in mouse cortices, but de‐ clines over time. By contrast, TLR2 expression appears around E15.5 and increases with time [70, 88]. Moreover, in standard in vitro neurosphere assays (used to assess developmental potential of NPCs), both TLR2 and TLR3 activation appear to regulate NPC proliferation. These studies raise questions regarding the expression of other TLRs during brain develop‐ ment, and how that expression pattern is altered when dams are exposed to TLR agonists, such as poly(I:C), during pregnancy. The structural and/or functional abnormalities seen in the brains of offspring from poly(I:C)-injected pregnant dams may correlate with alterations of the normal patterns of TLR expression in the developing brain. Therefore, disruption of the normal TLR expression pattern might be involved in the observed structural and/or functional changes in the brain of individuals with neurodevelopmental disorders, such as schizophrenia and autism.

these tissues for surface markers (Sca-1 and c-kit) that define HSCs, and the lineage-specific progenitors for T cells (TCR, CD3), B cells (sIg, CD19), and myeloid cells (CD11b, CD11c).

Pro-Inflammatory Phenotype Induced by Maternal Immune Stimulation During Pregnancy

http://dx.doi.org/10.5772/53990

125

An example of our results for HSCs in fetal liver is presented in **Table 3**. Pregnant dams were injected at E12 with either PBS or poly(I:C), and fetuses were examined 24 hrs later. The data show that in comparison to fetuses from PBS-injected dams, fetal livers from poly(I:C)-injected dams had a 4- to 5-fold increase in the percentage of HSCs that were double-positive for Sca-1 and c-kit, and almost a 3-fold increase in the percentage of HSCs that expressed only Sca-1, which are early **C**ommon **L**ymphoid **P**rogenitors (CLP). By contrast, the percent of HSCs that expressed only c-kit, which are early **C**ommon **M**yeloid **P**rogenitors (CMP), was similar in all fetal livers. These results are intriguing because they indicate hyper-proliferation of HSCs and early CLP, which may forecast the preferential changes we have observed in mature T lympho‐

In addition to our investigation of the consequences of maternal immune stimulation to preg‐ nant dams, embryonic tissues, and 2-4 wk old neonates, we have also extended our studies to adult offspring of poly(I:C)-injected (vs. PBS-injected). Our guiding hypothesis is that as a re‐ sult of in utero exposure of the fetus to cytokines elicited by maternal immune stimulation (act‐ ing as a "first hit"), developmental programming of the immune system occurs in offspring, which persists postnatally and into adulthood. In the case of this prenatal model, such fetal programming results in development of a "pro-inflammatory" phenotype, such that upon subsequent postnatal exposure to an immune stimulus (i.e., second hit) the offspring of poly(I:C)-injected pregnant dams exhibit exacerbated responses in comparison to offspring of PBS-injected dams. Such a scenario is also consistent with the "multiple hit" concept of mental disorders [104, 105]. In the context of ASD, this would mean that abnormalities of behavior and immune dysregulation in some children with ASD could reflect such developmental program‐ ming during embryonic development that is manifested postnatally upon encounter with a second hit to their immune system. We tested this hypothesis by using adult offspring of poly(I:C)-injected (vs. PBS-injected) pregnant dams in selected in vivo experimental models

*Inflammatory response to TLR2 agonist, zymosan:* We induced an antigen non-specific acute in‐ flammatory response in the peritoneal cavity with zymosan (TLR-2 agonist), and assessed

Adult offspring from immunologically naïve poly(I:C)-injected dams were injected i.p. with PBS (control) or zymosan. Adult offspring from immunologically naïve PBS-injected dams were also injected with PBS or zymosan for comparison. Mice were euthanized at 4 hrs, and 2ml of cold PBS was used to flush their peritoneal contents. The number and type of perito‐ neal exudate cells were determined by manual counting and FACS analysis, and the perito‐

the qualitative and quantitative nature of the inflammatory response 4 hrs later [106].

cytes in the adult offspring of poly(I:C)-injected dams [43-45, 60].

that involve activation of their innate and/or adaptive immune systems.

neal fluid was analyzed for the presence of cytokines.

**3.** *In vivo* **proof-of-concept experiments**


Pregnant dams were injected at E12 and fetuses were obtained 24 hrs later. Fetal liver cells from individual fetuses were analyzed by FACS for expression of markers that define HSCs and early common progenitor cells.

#### **Table 3.** Hematopoietic Stem Cells in Fetal Liver

*TLR expression on hematopoietic stem cells (HSCs):* As previously mentioned, maternal expo‐ sure to poly(I:C) during pregnancy induces production of pro-inflammatory cytokines, in‐ cluding significant increases in IL-6 in maternal circulation, amniotic fluid, placenta, and fetal brain [51, 89-93]. Direct injection of IL-6 to pregnant dams also results in consequences for the offspring, including structural abnormalities in the brain, as well as behavioral and cognitive abnormalities [30, 34-36, 38]. However, IL-6 also affects the immune system; it is an autocrine growth factor for thymic epithelial cells [94], stimulates fetal hematopoiesis [95], and can alter the balance of Tregs and Th17 cells towards the pro-inflammatory Th17 phenotype [96-101]. Thus, IL-6 is a key player in the differentiation of cells in the immune system, and may play a role in the immune dysregulation seen in ASD.

Recent studies have also revealed that HSCs not only respond to cytokine signaling to ini‐ tiate myelopoiesis and lymphopoiesis, but also can sense microbial pathogens directly via TLR signaling [78]. Administration of nanomolar concentrations of the TLR4 agonist, LPS, triggers emigration of monocytes from the BM into the bloodstream, indicating that circulat‐ ing levels of TLR ligands can also stimulate HSCs within hematopoietic tissues [102]. Addi‐ tionally, treatment of mice with TLR3 agonist poly(I:C) activates HSCs to proliferate [103]. Therefore, it is likely that in the prenatal model we are studying, HSCs are influenced not only by the poly(I:C) induced cytokines elicited during pregnancy, but also by this TLR3 ag‐ onist as well. Therefore, we have examined placentas, fetal livers, and neonatal bone mar‐ row from poly(I:C)-injected (vs.PBS-injected) pregnant dams and offspring to characterize the changes in HSCs, as well as lineage-specific progenitor cells. We examined cells from these tissues for surface markers (Sca-1 and c-kit) that define HSCs, and the lineage-specific progenitors for T cells (TCR, CD3), B cells (sIg, CD19), and myeloid cells (CD11b, CD11c).

An example of our results for HSCs in fetal liver is presented in **Table 3**. Pregnant dams were injected at E12 with either PBS or poly(I:C), and fetuses were examined 24 hrs later. The data show that in comparison to fetuses from PBS-injected dams, fetal livers from poly(I:C)-injected dams had a 4- to 5-fold increase in the percentage of HSCs that were double-positive for Sca-1 and c-kit, and almost a 3-fold increase in the percentage of HSCs that expressed only Sca-1, which are early **C**ommon **L**ymphoid **P**rogenitors (CLP). By contrast, the percent of HSCs that expressed only c-kit, which are early **C**ommon **M**yeloid **P**rogenitors (CMP), was similar in all fetal livers. These results are intriguing because they indicate hyper-proliferation of HSCs and early CLP, which may forecast the preferential changes we have observed in mature T lympho‐ cytes in the adult offspring of poly(I:C)-injected dams [43-45, 60].
