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

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

> **PBS-injected dams poly(I:C)-injected dams** #1 #2 #1 #2

**Cell Marker Percent Positive Cells in Fetuses from**

were analyzed by FACS for expression of markers that define HSCs and early common progenitor cells.

system, and may play a role in the immune dysregulation seen in ASD.

Sca-1+, c-kit+ (HSC) 0.3 0.3 1.5 1.3

, c-kit+ (CMP) 24.0 25.9 21.6 24.5 Sca-1+, c-kit- (CLP) 9.6 8.0 23.1 23.1

Pregnant dams were injected at E12 and fetuses were obtained 24 hrs later. Fetal liver cells from individual fetuses

*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

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

schizophrenia and autism.

124 Recent Advances in Autism Spectrum Disorders - Volume I

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

Sca-1-

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

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

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‐ neal fluid was analyzed for the presence of cytokines.

As shown in Table 4, the >2 fold increase in total Peritoneal Exudate Cell (PEC) count in the zymosan-injected poly(I:C) offspring was significantly higher than the count recovered from zymosan-injected PBS offspring. In contrast, there were no significant differences in absolute PEC numbers in control PBS-injected adult poly(I:C) or PBS offspring. The peritoneal cellu‐ lar infiltrate in offspring injected with PBS was primarily mononuclear cells (monocytes and lymphocytes) (Figure 8A). In contrast, the acute cellular inflammatory response in the peri‐ toneal cavity of zymosan-injected offspring was mostly neutrophils (Figure 8B).

The percentage of neutrophils in zymosan-injected offspring from PBS-injected and poly(I:C) injected offspring (Table 4) were similarly high (i.e., 70% and 81%, respectively). However, be‐ cause the total number of PEC recovered from zymosan-injected offspring from poly(I:C) injected (vs. PBS-injected) dams was significantly higher, the absolute number of neutrophils from zymosan-injected offspring was also significantly greater in offspring from poly(I:C)-in‐ jected dams. Given the huge infiltration of neutrophils into the peritoneal cavity in zymosaninjected offspring, we also examined the bone marrow for evidence of increased myeloid activity, and found evidence of increased myeloid activity in mice showing PEC counts in ex‐

in fluid obtained from the peritoneal cavity of zymosan-injected poly(I:C) offspring vs. PBS off‐ spring at 4 hrs after zymosan injection. Although not shown in the table, levels of TNF-α and

*Results from the myocardial Ischemia/reperfusion model:* Based on the results we obtained using injection of zymosan to mimic the acute inflammatory response induced by an infectious or‐ ganism, we wished to determine if the offspring of immunologically naïve poly(I:C)-injected dams would also mount a more robust inflammatory responses to endogenous molecules created by non-infectious tissue injury. The persistent neuroinflammation observed in brains of individuals with autism and in rodents from experimental models of neurodeve‐ lopmental disorders may be triggered by such endogenous stimuli. For these experiments, we selected a well-characterized cardiac model in which ischemia/reperfusion causes a "sterile" inflammatory response. After an acute myocardial infarction, reperfusion (by thrombolytic therapy or primary percutaneous intervention) is currently the most effective strategy to minimize myocardial damage and improve clinical outcome [107]. Paradoxically, restoring blood flow to the ischemic heart tissue can also induce injury – a phenomenon called myocardial reperfusion injury (R/I). The modes of myocardial cell injury and death following myocardial R/I are apoptosis, autophagy, and necrosis, and several underlying mechanisms have been identified or proposed [108-112]. However, one well-studied cause of myocardial R/I is the host inflammatory response that occurs during reperfusion. Despite the fact that ischemia and reperfusion takes place in a sterile environment, activation of in‐ nate and adaptive immune responses occurs and contributes to injury (reviewed in [112]). Contributing factors of reperfusion-induced inflammation include activation of Toll-like Re‐ ceptors (TLRs), complement activation, free radical generation, cytokine cascade initiated by release of pro-inflammatory cytokines, and chemokine upregulation [113-116]. The presence of these immune mediators leads to recruitment of neutrophils to the ischemic myocardium, which exert cytotoxic effects themselves by release of proteolytic enzymes. Another paradox of myocardial reperfusion is that it may also significantly enhance a healing process. Studies have shown that Monocyte Chemoattractant Protein-1 (MCP-1) is also induced in the in‐ farcted area, which may regulate myeloid cell recruitment, leading to accumulation of mac‐ rophages and mast cells that secrete angiogenesis-stimulating factors, which facilitate

For these experiments, adult offspring of immunologically naïve poly(I:C)-injected and PBSinjected dams were anesthetized, intubated and ventilated; the heart was exposed by a thor‐

IL-10 were also significantly higher in these zymosan-injected poly(I:C) offspring.

cells. As also shown in Table 4, significantly higher levels of IL-6 were observed

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cess of 10 x 106

myocardial repair [117, 118].


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 compari‐ son. Mice were euthanized at 4 hrs, and 2ml of cold PBS was used to flush their peritoneal contents. The number and type of peritoneal exudate cells (PEC) were determined by manual counting and FACS analysis, and sera and perito‐ neal fluids were analyzed for the presence of cytokines. N = 5-8, \*\* P=0.016 (student's t-test). \*P< 0.05 (student's ttest)

**Table 4.** Zymosan-induced acute inflammatory responses in offspring

**Figure 8. Acute inflammatory response in zymosan-injected adult offspring.** Adult offspring from poly(I:C)-inject‐ ed non-immune dams were injected i.p. with 1 ml of zymosan suspension or PBS (control). Four hours after injection, peritoneal cavities were flushed with ice cold PBS. Cytospin slides were made from peritoneal exudate cells, and stained with Wright's/Giemsa stain. (A) PBS-injected offspring from poly(I:C)-injected non-immune dams at 630X. (B) zymosan-injected offspring from poly(I:C)-injected non-immune dams at 630X.

The percentage of neutrophils in zymosan-injected offspring from PBS-injected and poly(I:C) injected offspring (Table 4) were similarly high (i.e., 70% and 81%, respectively). However, be‐ cause the total number of PEC recovered from zymosan-injected offspring from poly(I:C) injected (vs. PBS-injected) dams was significantly higher, the absolute number of neutrophils from zymosan-injected offspring was also significantly greater in offspring from poly(I:C)-in‐ jected dams. Given the huge infiltration of neutrophils into the peritoneal cavity in zymosaninjected offspring, we also examined the bone marrow for evidence of increased myeloid activity, and found evidence of increased myeloid activity in mice showing PEC counts in ex‐ cess of 10 x 106 cells. As also shown in Table 4, significantly higher levels of IL-6 were observed in fluid obtained from the peritoneal cavity of zymosan-injected poly(I:C) offspring vs. PBS off‐ spring at 4 hrs after zymosan injection. Although not shown in the table, levels of TNF-α and IL-10 were also significantly higher in these zymosan-injected poly(I:C) offspring.

As shown in Table 4, the >2 fold increase in total Peritoneal Exudate Cell (PEC) count in the zymosan-injected poly(I:C) offspring was significantly higher than the count recovered from zymosan-injected PBS offspring. In contrast, there were no significant differences in absolute PEC numbers in control PBS-injected adult poly(I:C) or PBS offspring. The peritoneal cellu‐ lar infiltrate in offspring injected with PBS was primarily mononuclear cells (monocytes and lymphocytes) (Figure 8A). In contrast, the acute cellular inflammatory response in the peri‐

**Neutrophils IL-6 (pg/ml)**

**Sera Peritoneal Fluid**

**Absolute number (x106)**

Zymosan 6.3 ± 1.8 70.0 ± 10 4.40 ± 1.9 420 ± 200 1176 ± 586

PBS 1.0 ± 0.4 <5.0 <0.005 8 ± 2.7 6 ± 2.5 Zymosan 13.3 ± 2.1\*\* 81.0 ± 6 10.8 ± 2.0\*\* 2692 ± 514\* 7808 ± 1306\*

toneal cavity of zymosan-injected offspring was mostly neutrophils (Figure 8B).

PBS-injected dams PBS 0.8 ± 0.3 <5.0 <0.004 4 ± 0.6 15 ± 7.9

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 compari‐ son. Mice were euthanized at 4 hrs, and 2ml of cold PBS was used to flush their peritoneal contents. The number and type of peritoneal exudate cells (PEC) were determined by manual counting and FACS analysis, and sera and perito‐ neal fluids were analyzed for the presence of cytokines. N = 5-8, \*\* P=0.016 (student's t-test). \*P< 0.05 (student's t-

**Figure 8. Acute inflammatory response in zymosan-injected adult offspring.** Adult offspring from poly(I:C)-inject‐ ed non-immune dams were injected i.p. with 1 ml of zymosan suspension or PBS (control). Four hours after injection, peritoneal cavities were flushed with ice cold PBS. Cytospin slides were made from peritoneal exudate cells, and stained with Wright's/Giemsa stain. (A) PBS-injected offspring from poly(I:C)-injected non-immune dams at 630X. (B)

**Total PEC (x106)**

**From Injected with Percent**

**Table 4.** Zymosan-induced acute inflammatory responses in offspring

zymosan-injected offspring from poly(I:C)-injected non-immune dams at 630X.

**Offspring**

126 Recent Advances in Autism Spectrum Disorders - Volume I

Poly(I:C)-injected dams

test)

*Results from the myocardial Ischemia/reperfusion model:* Based on the results we obtained using injection of zymosan to mimic the acute inflammatory response induced by an infectious or‐ ganism, we wished to determine if the offspring of immunologically naïve poly(I:C)-injected dams would also mount a more robust inflammatory responses to endogenous molecules created by non-infectious tissue injury. The persistent neuroinflammation observed in brains of individuals with autism and in rodents from experimental models of neurodeve‐ lopmental disorders may be triggered by such endogenous stimuli. For these experiments, we selected a well-characterized cardiac model in which ischemia/reperfusion causes a "sterile" inflammatory response. After an acute myocardial infarction, reperfusion (by thrombolytic therapy or primary percutaneous intervention) is currently the most effective strategy to minimize myocardial damage and improve clinical outcome [107]. Paradoxically, restoring blood flow to the ischemic heart tissue can also induce injury – a phenomenon called myocardial reperfusion injury (R/I). The modes of myocardial cell injury and death following myocardial R/I are apoptosis, autophagy, and necrosis, and several underlying mechanisms have been identified or proposed [108-112]. However, one well-studied cause of myocardial R/I is the host inflammatory response that occurs during reperfusion. Despite the fact that ischemia and reperfusion takes place in a sterile environment, activation of in‐ nate and adaptive immune responses occurs and contributes to injury (reviewed in [112]). Contributing factors of reperfusion-induced inflammation include activation of Toll-like Re‐ ceptors (TLRs), complement activation, free radical generation, cytokine cascade initiated by release of pro-inflammatory cytokines, and chemokine upregulation [113-116]. The presence of these immune mediators leads to recruitment of neutrophils to the ischemic myocardium, which exert cytotoxic effects themselves by release of proteolytic enzymes. Another paradox of myocardial reperfusion is that it may also significantly enhance a healing process. Studies have shown that Monocyte Chemoattractant Protein-1 (MCP-1) is also induced in the in‐ farcted area, which may regulate myeloid cell recruitment, leading to accumulation of mac‐ rophages and mast cells that secrete angiogenesis-stimulating factors, which facilitate myocardial repair [117, 118].

For these experiments, adult offspring of immunologically naïve poly(I:C)-injected and PBSinjected dams were anesthetized, intubated and ventilated; the heart was exposed by a thor‐ acotomy through the 4th and 5th ribs, and a suture was passed under the left coronary artery. The left coronary artery was occluded for a period of 20 min, and reperfusion applied for 24 hrs. Reperfusion was achieved by removal of the occlusion, the thoracotomy incision was closed, and mice were allowed to recover under monitoring in an incubator. After 24 hr of reperfusion, mice were assessed for cardiac injury as previously described [119, 120].

Encephalomyelitis (EAE), a mouse model of multiple sclerosis [122-128]. Female offspring of poly(I:C) (vs. PBS) -injected pregnant dams were injected s.c. in each hind flank with an ence‐ phalogenic-peptide (MOG35-55) in Complete Freund 's adjuvant (CFA). I.p. injections of pertus‐ sis toxin were given after MOG immunization to enhance the immune response and promote T cell migration into the brain [129]. Typically, 10 – 12 days after injection of MOG and pertussis, 90% of B6 mice develop progressively: weakness and paralysis in their tail, hindlimb paresis and finally hindlimb paralysis. Controls that receive CFA and pertussis toxin, but no MOG, do

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However, offspring of poly(I:C)-injected immune pregnant dams exhibited clinical signs of EAE significantly earlier and with higher frequency than offspring of poly(I:C)-injected nonimmune dams (Figure 10). More than 70% of poly(I:C) immune offspring began to show clinical signs of EAE by day 4 after MOG35-55 immunization compared to none seen in poly(I:C) non-immune offspring. On day 7 after immunization, 25% of poly(I:C) non-im‐ mune offspring began to show symptoms, but this was still significantly lower than the >70% seen in poly(I:C) immune offspring. By day 9, >60% of poly(I:C) non-immune off‐ spring showed clinical signs of EAE. In addition to the higher frequency of clinical signs of EAE, poly(I:C) immune offspring also had significantly higher disease severity from days 4-7 post MOG35-55 immunization compared to poly(I:C) non-immune offspring [45, 60]. From days 9-20, the development of EAE among offspring was very similar in both groups. How‐ ever, the earlier appearance of clinical symptoms affords a window of opportunity to inves‐ tigate underlying mechanisms in the EAE model that can be applied in future studies of mechanisms of neuroinflammation and pathogenesis in experimental models of autism.

**Figure 10. Frequency of mice showing clinical signs of EAE.** Adult offspring of of poly(I:C)-injected dams with im‐ munological memory [vs. immunologically naïve poly(I:C)-injected dams] were injected s.c. with MOG in CFA and i.p. with pertussis toxin. Mice were scored for clinical signs of neurological impairment, and the percent of mice showing

clinical signs at the indicated times after immunization is shown in this figure.

not develop clinical signs of EAE.

As shown in Figure 9, significantly greater cardiac damage was observed in offspring from immunologically naïve poly(I:C)-injected dams than in offspring from control PBS-injected dams. We are currently assessing the underlying mechanisms responsible for the difference in levels of damage in experimental and control offspring. Myocardial I/R induces infiltra‐ tion of inflammatory cells, such as neutrophils that secrete cytokines/chemokines, including IL-6 and TNFα, which in turn contribute to cell death, fibrosis and reduced myocardial con‐ tractility [121]. Therefore, it is likely that similar underlying inflammatory mechanisms also occur in the myocardial I/R model as those described above for the acute inflammation in‐ duced by zymosan. In the zymosan and myocardial I/R models, the response is measured within hours of the immune stimulus. This indicates that elements of the innate immune system are the primary mediators of the pathology, and suggest that modification of these components has occurred as a result of maternal immune stimulation during pregnancy.

**Figure 9. Cardiac damage in offspring of poly(I:C)-injected vs. PBS-injected immunologically naïve pregnant dams.** Adult offspring from poly(I:C)-injected (Exp) and PBS-injected (Control) dams were subjected to cardiac ische‐ mia (20 min) and reperfusion (24 hrs), and assessed for cardiac damage (indicated by infarct size). Offspring from poly(I:C)-injected dams exhibited significantly greater cardiac damage (p=0.011; student's t-test) than control off‐ spring. (N = 8 mice/group; 8 weeks of age)

*Response to auto-antigens*: In order determine if components of the adaptive immune system al‐ so mount more robust responses following immune stimulation we chose a well-characterized EAE model of an immune-mediated disease, and used adult offspring of pregnant dams with immunological memory. We have previously shown that T cells from offspring of poly(I:C)-in‐ jected pregnant dams with immunological memory (i.e., immune) preferentially differentiate to become Th17 cells after in vitro activation [43-45, 60]. In contrast, T cells from offspring of poly(I:C)-injected immunologically naïve pregnant dams (i.e., non-immune) do not show such Th17 cell preferential differentiation. Th17 cells have been shown to be involved in the neuro‐ pathology responsible for the clinical symptoms that develop in Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of multiple sclerosis [122-128]. Female offspring of poly(I:C) (vs. PBS) -injected pregnant dams were injected s.c. in each hind flank with an ence‐ phalogenic-peptide (MOG35-55) in Complete Freund 's adjuvant (CFA). I.p. injections of pertus‐ sis toxin were given after MOG immunization to enhance the immune response and promote T cell migration into the brain [129]. Typically, 10 – 12 days after injection of MOG and pertussis, 90% of B6 mice develop progressively: weakness and paralysis in their tail, hindlimb paresis and finally hindlimb paralysis. Controls that receive CFA and pertussis toxin, but no MOG, do not develop clinical signs of EAE.

acotomy through the 4th and 5th ribs, and a suture was passed under the left coronary artery. The left coronary artery was occluded for a period of 20 min, and reperfusion applied for 24 hrs. Reperfusion was achieved by removal of the occlusion, the thoracotomy incision was closed, and mice were allowed to recover under monitoring in an incubator. After 24 hr of reperfusion, mice were assessed for cardiac injury as previously described [119, 120].

As shown in Figure 9, significantly greater cardiac damage was observed in offspring from immunologically naïve poly(I:C)-injected dams than in offspring from control PBS-injected dams. We are currently assessing the underlying mechanisms responsible for the difference in levels of damage in experimental and control offspring. Myocardial I/R induces infiltra‐ tion of inflammatory cells, such as neutrophils that secrete cytokines/chemokines, including IL-6 and TNFα, which in turn contribute to cell death, fibrosis and reduced myocardial con‐ tractility [121]. Therefore, it is likely that similar underlying inflammatory mechanisms also occur in the myocardial I/R model as those described above for the acute inflammation in‐ duced by zymosan. In the zymosan and myocardial I/R models, the response is measured within hours of the immune stimulus. This indicates that elements of the innate immune system are the primary mediators of the pathology, and suggest that modification of these components has occurred as a result of maternal immune stimulation during pregnancy.

**Figure 9. Cardiac damage in offspring of poly(I:C)-injected vs. PBS-injected immunologically naïve pregnant dams.** Adult offspring from poly(I:C)-injected (Exp) and PBS-injected (Control) dams were subjected to cardiac ische‐ mia (20 min) and reperfusion (24 hrs), and assessed for cardiac damage (indicated by infarct size). Offspring from poly(I:C)-injected dams exhibited significantly greater cardiac damage (p=0.011; student's t-test) than control off‐

*Response to auto-antigens*: In order determine if components of the adaptive immune system al‐ so mount more robust responses following immune stimulation we chose a well-characterized EAE model of an immune-mediated disease, and used adult offspring of pregnant dams with immunological memory. We have previously shown that T cells from offspring of poly(I:C)-in‐ jected pregnant dams with immunological memory (i.e., immune) preferentially differentiate to become Th17 cells after in vitro activation [43-45, 60]. In contrast, T cells from offspring of poly(I:C)-injected immunologically naïve pregnant dams (i.e., non-immune) do not show such Th17 cell preferential differentiation. Th17 cells have been shown to be involved in the neuro‐ pathology responsible for the clinical symptoms that develop in Experimental Autoimmune

spring. (N = 8 mice/group; 8 weeks of age)

128 Recent Advances in Autism Spectrum Disorders - Volume I

However, offspring of poly(I:C)-injected immune pregnant dams exhibited clinical signs of EAE significantly earlier and with higher frequency than offspring of poly(I:C)-injected nonimmune dams (Figure 10). More than 70% of poly(I:C) immune offspring began to show clinical signs of EAE by day 4 after MOG35-55 immunization compared to none seen in poly(I:C) non-immune offspring. On day 7 after immunization, 25% of poly(I:C) non-im‐ mune offspring began to show symptoms, but this was still significantly lower than the >70% seen in poly(I:C) immune offspring. By day 9, >60% of poly(I:C) non-immune off‐ spring showed clinical signs of EAE. In addition to the higher frequency of clinical signs of EAE, poly(I:C) immune offspring also had significantly higher disease severity from days 4-7 post MOG35-55 immunization compared to poly(I:C) non-immune offspring [45, 60]. From days 9-20, the development of EAE among offspring was very similar in both groups. How‐ ever, the earlier appearance of clinical symptoms affords a window of opportunity to inves‐ tigate underlying mechanisms in the EAE model that can be applied in future studies of mechanisms of neuroinflammation and pathogenesis in experimental models of autism.

**Figure 10. Frequency of mice showing clinical signs of EAE.** Adult offspring of of poly(I:C)-injected dams with im‐ munological memory [vs. immunologically naïve poly(I:C)-injected dams] were injected s.c. with MOG in CFA and i.p. with pertussis toxin. Mice were scored for clinical signs of neurological impairment, and the percent of mice showing clinical signs at the indicated times after immunization is shown in this figure.

These results are consistent with our hypothesis of fetal programming due to effects of ma‐ ternal immune stimulation during pregnancy, leading to increased susceptibility of off‐ spring to a "second hit" postnatal stimulus. This is likely due to an overall heightened immune responsiveness to develop EAE by MOG-specific Th cells that preferentially differ‐ entiate to become Th17 cells in these pro-inflammatory mice and/or a lower antigen thresh‐ old for initiation of an immune response. Another contributing factor could be differential responses of these pro-inflammatory offspring to TLR agonists on the mycobacteria in CFA (TLR2) and pertussis toxin (TLR4) used as part of the MOG immunization protocol [130, 131]. Support for this possibility is shown in Figure 7, where offspring of poly(I:C)-injected dams who possess immunological memory showed >3-fold higher expression of TLR2 and TLR4 compared to controls.

kines and T helper (Th) lymphocyte subsets. Our investigation of this mouse model has also provided a scientific basis for an ongoing translational research project to determine if similar molecular pathogenic mechanisms are involved in the cohort of ASD children who also exhibit evidence of immune dysregulation. Thus, mothers of autistic children in this cohort have polymorphisms in the same cytokine genes that promote inflammatory reactions in our mouse model, and their children with autism and immune dysregula‐

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Convincing evidence from this model has shown that pro-inflammatory cytokines pro‐ duced by maternal immune stimulation during pregnancy induce changes in the devel‐ opment of the immune system and brain of offspring that result in similar immunological and behavioral manifestations as those seen in individuals with ASD. Therefore, our results are relevant to the concept of developmental programming of the immune system. In utero exposure to these cytokines produces offspring that exhibit a pro-inflammatory phenotype, which persists throughout the neonatal period and into adulthood. Subsequently, upon postnatal exposure to agents that stimulate the immune system, offspring that exhibit this phenotype mount a more robust immune response in which pro-inflammatory immune elements (i.e., Th17 cells and cytokines) predominate. Th17 cells have been shown to mediate immunopathology in numerous disorders that model human diseases, such as multiple sclerosis, arthritis, inflammatory bowel disease, atherosclerosis, and diabetes. Our use of offspring that have Th cells with the potential to preferentially differentiate into Th17 cells will also determine the contribution of Th17

The nature and timing of this second hit to the immune system may also be a critical de‐ termining factor in the manifestation of immune outcomes. Thus, if immune stimulation occurs very early in life when organ systems, such as the brain, are still developing, it may lead to neurodevelopmental disorders like ASD. Contrastingly, if the second hit oc‐ curs later in life, the outcome may be manifested as an autoimmune disorder. However, possession of a pro-inflammatory phenotype as described in our model is not necessarily a disadvantage. In certain clinical scenarios, such as malignancy or infection with patho‐ genic micro-organisms, a more robust immune response may provide survival advant‐ age. Indeed, our preliminary results in an infection model indicate that the offspring of poly(I:C)-injected pregnant dams that exhibit a pro-inflammatory phenotype show in‐ creased survival time and lower pathogen burden than control offspring from PBS-inject‐

As with many other components of the immune system, the effector functions resulting from developmental programming induced by maternal immune stimulation during preg‐ nancy have the potential to be a double-edged sword with outcomes that can be either detri‐ mental or beneficial. The future challenge in studying this prenatal model system will be to sufficiently understand the underlying cellular and molecular mechanisms to enable the de‐ sign of effective therapeutic interventions to inhibit outcomes that are harmful, and enhance

tion inherit the maternal pro-inflammatory phenotype.

cells to the etiology and pathogenesis of ASD.

ed pregnant dams.

those that are beneficial.

Overall, our results are consistent with the concept of developmental programming of the immune system [71, 132-136]. These changes persist into adulthood, and increase the vulner‐ ability of offspring from poly(I:C)-injected dams to develop immune-mediated diseases when exposed to subsequent antigen specific, as well as antigen non-specific immune chal‐ lenges. There is considerable plasticity of the developing immune system, and maternal stressors, such as immune stimulation during pregnancy, can modulate normal develop‐ ment [137]. Immune stimuli during the perinatal period of life can also act as a vulnerability factor for later-life alterations of immune responsiveness [132]. Such fetal programming has been described in relation to abnormalities of metabolism, growth, and behavior in offspring [138-140], as well as in relation to allergic and autoimmune disorders [133-135, 141-143]. The fetal programming of the developing immune system in this prenatal mouse model descri‐ bed herein is most likely mediated by cytokines and/or other inflammatory mediators pro‐ duced by immune stimulation in response to poly(I:C) given to the pregnant dam. However, as we have previously shown, the sources of these products of immune stimulation are of both maternal and fetal origin [44].

### **4. Summary and conclusions**

The results from our investigation of the poly(I:C)-induced prenatal model of neurodeve‐ lopmental disorders further identifies and characterizes gene-environment interactions (i.e., maternal immune response genes vs. environmental antigens) that influence fetal development in ways that have consequences for health and disease of offspring. Further characterization of this model presents excellent opportunities to define the underlying mechanisms responsible for the alterations that occur during embryological development, which persist and are manifested in adult offspring. We are using this model to examine the peripheral immune system of offspring to identify mechanisms that explain the im‐ mune dysregulation that is characteristic in a significant cohort of children with Autism Spectrum Disorders (ASD). The immunological changes we find in offspring of dams that receive immune stimulation during pregnancy involve significant differences in cyto‐ kines and T helper (Th) lymphocyte subsets. Our investigation of this mouse model has also provided a scientific basis for an ongoing translational research project to determine if similar molecular pathogenic mechanisms are involved in the cohort of ASD children who also exhibit evidence of immune dysregulation. Thus, mothers of autistic children in this cohort have polymorphisms in the same cytokine genes that promote inflammatory reactions in our mouse model, and their children with autism and immune dysregula‐ tion inherit the maternal pro-inflammatory phenotype.

These results are consistent with our hypothesis of fetal programming due to effects of ma‐ ternal immune stimulation during pregnancy, leading to increased susceptibility of off‐ spring to a "second hit" postnatal stimulus. This is likely due to an overall heightened immune responsiveness to develop EAE by MOG-specific Th cells that preferentially differ‐ entiate to become Th17 cells in these pro-inflammatory mice and/or a lower antigen thresh‐ old for initiation of an immune response. Another contributing factor could be differential responses of these pro-inflammatory offspring to TLR agonists on the mycobacteria in CFA (TLR2) and pertussis toxin (TLR4) used as part of the MOG immunization protocol [130, 131]. Support for this possibility is shown in Figure 7, where offspring of poly(I:C)-injected dams who possess immunological memory showed >3-fold higher expression of TLR2 and

Overall, our results are consistent with the concept of developmental programming of the immune system [71, 132-136]. These changes persist into adulthood, and increase the vulner‐ ability of offspring from poly(I:C)-injected dams to develop immune-mediated diseases when exposed to subsequent antigen specific, as well as antigen non-specific immune chal‐ lenges. There is considerable plasticity of the developing immune system, and maternal stressors, such as immune stimulation during pregnancy, can modulate normal develop‐ ment [137]. Immune stimuli during the perinatal period of life can also act as a vulnerability factor for later-life alterations of immune responsiveness [132]. Such fetal programming has been described in relation to abnormalities of metabolism, growth, and behavior in offspring [138-140], as well as in relation to allergic and autoimmune disorders [133-135, 141-143]. The fetal programming of the developing immune system in this prenatal mouse model descri‐ bed herein is most likely mediated by cytokines and/or other inflammatory mediators pro‐ duced by immune stimulation in response to poly(I:C) given to the pregnant dam. However, as we have previously shown, the sources of these products of immune stimulation are of

The results from our investigation of the poly(I:C)-induced prenatal model of neurodeve‐ lopmental disorders further identifies and characterizes gene-environment interactions (i.e., maternal immune response genes vs. environmental antigens) that influence fetal development in ways that have consequences for health and disease of offspring. Further characterization of this model presents excellent opportunities to define the underlying mechanisms responsible for the alterations that occur during embryological development, which persist and are manifested in adult offspring. We are using this model to examine the peripheral immune system of offspring to identify mechanisms that explain the im‐ mune dysregulation that is characteristic in a significant cohort of children with Autism Spectrum Disorders (ASD). The immunological changes we find in offspring of dams that receive immune stimulation during pregnancy involve significant differences in cyto‐

TLR4 compared to controls.

130 Recent Advances in Autism Spectrum Disorders - Volume I

both maternal and fetal origin [44].

**4. Summary and conclusions**

Convincing evidence from this model has shown that pro-inflammatory cytokines pro‐ duced by maternal immune stimulation during pregnancy induce changes in the devel‐ opment of the immune system and brain of offspring that result in similar immunological and behavioral manifestations as those seen in individuals with ASD. Therefore, our results are relevant to the concept of developmental programming of the immune system. In utero exposure to these cytokines produces offspring that exhibit a pro-inflammatory phenotype, which persists throughout the neonatal period and into adulthood. Subsequently, upon postnatal exposure to agents that stimulate the immune system, offspring that exhibit this phenotype mount a more robust immune response in which pro-inflammatory immune elements (i.e., Th17 cells and cytokines) predominate. Th17 cells have been shown to mediate immunopathology in numerous disorders that model human diseases, such as multiple sclerosis, arthritis, inflammatory bowel disease, atherosclerosis, and diabetes. Our use of offspring that have Th cells with the potential to preferentially differentiate into Th17 cells will also determine the contribution of Th17 cells to the etiology and pathogenesis of ASD.

The nature and timing of this second hit to the immune system may also be a critical de‐ termining factor in the manifestation of immune outcomes. Thus, if immune stimulation occurs very early in life when organ systems, such as the brain, are still developing, it may lead to neurodevelopmental disorders like ASD. Contrastingly, if the second hit oc‐ curs later in life, the outcome may be manifested as an autoimmune disorder. However, possession of a pro-inflammatory phenotype as described in our model is not necessarily a disadvantage. In certain clinical scenarios, such as malignancy or infection with patho‐ genic micro-organisms, a more robust immune response may provide survival advant‐ age. Indeed, our preliminary results in an infection model indicate that the offspring of poly(I:C)-injected pregnant dams that exhibit a pro-inflammatory phenotype show in‐ creased survival time and lower pathogen burden than control offspring from PBS-inject‐ ed pregnant dams.

As with many other components of the immune system, the effector functions resulting from developmental programming induced by maternal immune stimulation during preg‐ nancy have the potential to be a double-edged sword with outcomes that can be either detri‐ mental or beneficial. The future challenge in studying this prenatal model system will be to sufficiently understand the underlying cellular and molecular mechanisms to enable the de‐ sign of effective therapeutic interventions to inhibit outcomes that are harmful, and enhance those that are beneficial.
