Maternal Poly (I:C)-Induced Placental Inflammation and Endocrine Dysfunction Are Associated with Disrupted Corticogenesis in Mouse Offspring

TL;DR

Maternal inflammation during pregnancy disrupts placental function and leads to abnormal brain development in mouse offspring, potentially contributing to autism-like behaviors.

Contribution

This study identifies placental inflammation and endocrine dysfunction as key mechanisms linking maternal immune activation to disrupted neurogenesis in offspring.

Findings

Maternal poly(I:C) exposure increases placental monocytes and neutrophils, along with inflammatory cytokines.

Reduced placental NGF levels and sustained microglial activation are observed in offspring brains.

Altered cortical neurogenesis includes increased early-born and later-born neurons in offspring.

Abstract

Background/Objectives: Maternal immune activation (MIA) increases the risk of Autism Spectrum Disorders (ASD). Experimental models demonstrate that maternal exposure to bacterial endotoxin or the viral mimic polyinosinic:polycytidylic acid [poly (I:C)] reliably recapitulates ASD-like behavioral abnormalities in offspring, yet the underlying neurobiological mechanisms linking MIA to altered neurodevelopment remain incompletely understood. Increasing evidence highlights the placenta as a critical mediator in shaping fetal brain development through immunological and hormonal regulation. Likewise, disruption of placental regulatory functions upon MIA may therefore represent a mechanistic pathway. Here, we investigated how alterations in placental cytokine profiles, innate immune cell composition, and endocrine outputs relate to neuroinflammation and neurogenesis in the offspring. Methods: Pregnant mice at gestational day 12.5 received a single intraperitoneal injection of poly (I:C). Placental macrophages, neutrophils, inflammatory cytokines, and nerve growth factor (NGF) expression were examined 72 h later. Neurodevelopmental outcomes, including microglial activity and neurogenic markers, were evaluated in mouse offspring at postnatal day (P) 1 and 6. Results: MIA induced a significant accumulation of monocytes and neutrophils in the placenta, which was associated with elevated levels of a broad spectrum of inflammatory mediators, including Th17-biased proinflammatory cytokines, chemokines, and adhesion proteins, in the placenta and amniotic fluid. In contrast, the placenta-derived NGF levels were significantly reduced. MIA induced strong and sustained microglial activation in the fetal and neonatal brain. This inflammatory milieu was accompanied by disrupted cortical neurogenesis, characterized by a marked increase in Ki67+ neuronal progenitor cells (NPCs) in the subventricular zone (SVZ), overproduction of early-born Tbr1+ neurons at P1, later-born Satb2+ neurons at P6. Conclusions: Collectively, these findings suggest that heightened Th17 inflammatory signaling, coupled with impaired placental endocrine function, contributes to dysregulated cortical neurogenesis in the offspring.

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1. Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disability defined by impairments in social interaction and communication, accompanied by repetitive patterns of behavior or restricted interests. The etiology of ASD is multifactorial, involving complex interactions between diverse genetic susceptibilities and environmental influences. Among environmental risks, maternal infection is one of the most consistently associated ASD risk factors. Experimental work using the maternal immune activation (MIA) model has provided key mechanistic insight, offering strong evidence that maternal inflammatory signaling can causally disrupt fetal brain development and increase the likelihood of ASD-relevant neurodevelopmental outcomes. For instance, maternal exposure to either bacterial endotoxin lipopolysaccharide (LPS) or the viral mimic polyinosinic:polycytidylic acid [(poly I:C)] is sufficient to induce core behavioral features of ASD [1,2,3], although the molecular mechanisms through which MIA perturbs fetal brain development remain incompletely understood.

Inflammatory cytokines, especially interleukin-6 (IL-6) and its downstream IL-17a and retinoic acid receptor-related orphan receptor gamma t (RORγt), have been implicated as key mediators of aberrant neurodevelopment in MIA models [4,5]. Upon immune activation, these cytokines may originate from multiple sources, including the maternal, fetal, or placental compartments. The placenta is an angiogenic organ situated at the maternal-fetal interface, where it plays a critical role in regulating fetal neurodevelopment by providing immunomodulatory and endocrine support [6,7]. For instance, serotonin—a classical neurotransmitter that functions as a patterning molecule during early brain development, is synthesized primarily by the placenta before the emergence of raphe serotonergic neurons [8]. Likewise, several neurotrophic factors and hormones, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), are also produced by the placenta. Disruption of placental endocrine function is highlighted by the finding that MIA induced by poly (I:C) increases placental serotonin synthesis and blunts endogenous serotonergic axonal outgrowth in mice [9].

Disruption in cortical neurogenesis plays a putative role in ASD neuropathology, especially if the affected neurons and the associated connections are involved in language and communication domains. Given that neuronal progenitor cells (NPCs) derived from inducible pluripotent stem cells (iPSCs) of living ASD subjects exhibit accelerated proliferation potentials [10], while MIA enhances the proliferation of neural stem cells (NSCs) and increases intermediate neural progenitors in mouse offspring [11,12,13], there is a strong case that neurogenesis is disrupted in early fetal development. Therefore, this study was designed to test whether placental inflammation is associated with disrupted cortical development following maternal poly (I:C) exposure.

2. Experimental Procedures

2.1. Animals and Treatments

All animal experiments were performed in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Measures were taken throughout the study to reduce animal distress and discomfort. Male and female C57BL/6J mice (8 weeks of age; Jackson Laboratory, Bar Harbor, ME, USA) were maintained in same-sex groups of three to four per cage in a temperature- and humidity-regulated vivarium under a 12 h light/dark cycle (lights on at 8:00 a.m. and off at 8:00 p.m.), with unrestricted access to food and water. Following a two-week acclimation period, mice were paired for breeding at 10 weeks of age, and detection of a vaginal plug was defined as embryonic day 0.5 (E0.5). On E12.5, pregnant mice received an intraperitoneal injection of poly (I:C) (25 mg/kg body weight, MilliporeSigma, St. Louis, MO, USA, cat#P1530) or an equivalent volume of sterile saline as controls. Studies have previously shown that poly (I:C) at ~25 mg/kg could produce ASD-like neuropathology and behaviors in the mouse offspring [3,7,14]. All injections were administered at 4:00 p.m. local time. Body weight was recorded daily at 9:00 a.m. The experimental workflow is illustrated in Figure 1. A total of 24 pregnant mice were used in this study. We used an equal number of male and female mouse offspring for postnatal experiments. The sample size (n) indicated in the flow chart represents the number of independent litters. To control for the litter effect, only one representative offspring (matched for sex) was randomly selected from each litter for use in any particular IHC or WB experiment. Thus, n = 5–6 litters below signifies data from 5 to 6 individual pups, each from a different litter. For the placental cytokine array, only 1 litter was used, with all the placentas pooled for that particular litter.

2.2. Reagents

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other reagents were obtained from the following sources: poly (I:C) (MilliporeSigma, St. Louis, MO, USA), cytokine array kits (Cat#AAM-CYT-1000, RayBiotech, Peachtree Corners, GA, USA), and Western blot reagents (Bio-Rad Laboratories, Hercules, CA, USA). Details of antibodies are listed in Table 1.

2.3. Immunohistochemistry

At E15.5, the dams were deeply anesthetized using inhaled isoflurane. Placentas were carefully excised from the uterus, rinsed in saline, and fixed in 4% paraformaldehyde (PFA). Post-fixation, cryoprotection, sectioning, and immunohistochemistry procedures for the placenta were performed according to the protocols described for the brain. For neonatal brain tissue collection, offspring were anesthetized with inhaled isoflurane and transcardially perfused with saline, followed by 4% PFA. Brains were removed and post-fixed in 4% PFA for an additional 24 h, then cryoprotected by sequential incubation in 10%, 20%, and 30% sucrose solutions (24 h per step). Coronal brain sections (35 µm thick) were cut as free-floating sections using a freezing microtome (Leica SM 2000R, Wetzlar, Germany). For immunofluorescence, an antigen retrieval step was performed to enhance the detection of nuclear antigens. Sections were washed in phosphate-buffered saline (PBS) and transferred to 50 mL conical tubes containing citrate buffer (pH 6.0). Samples were heated in a water bath at 85 °C for 20 min, allowed to cool to room temperature (RT), and rinsed thoroughly with PBS. Non-specific binding was blocked by incubating sections for 1 h at RT in PBS containing 0.3% triton X-100 and 10% normal goat serum (Millipore, Billerica, MA, USA). Sections were then incubated overnight at 4 °C with primary antibodies diluted in blocking solution containing 0.3% triton under gentle agitation. The following day, sections were washed three times in PBS and incubated for 2 h at RT with appropriate fluorophore-conjugated secondary antibodies (Alexa Fluor 488, 1:400; Alexa Fluor 555, 1:2000. ThermoFisher Scientific, Waltham, MA, USA) diluted in blocking buffer. After final PBS washes, sections were mounted onto glass slides, air-dried, and coverslipped using mounting medium containing DAPI (100 nM) for nuclear counterstaining.

2.4. Imaging and Cell Counting

Fluorescence imaging was performed using a cooled monochrome camera mounted on a motorized fluorescence microscope (Nikon NIE, Nikon Instruments Inc., Melville, NY, USA). F4/80+ and CD68+ macrophages, as well as Ly6G+ neutrophils, were counted in the placental labyrinth. Iba1+ microglia were counted in the hippocampal dentate gyrus (DG). T-box brain transcription factor 1 (Tbr1)+, Special AT-rich sequence-binding protein 2 (Satb2)+, and Coup-TFI protein 2 (Ctip2)+ immature neurons were counted in the frontal cortex. Ki67+ NPCs were counted in the subventricular zone (SVZ) and DG. Briefly, the regions of interest (ROIs) were first delineated at 10× magnification under the DAPI channel, followed by acquisition of three random 40× images within the ROIs. Three consecutive longitudinal sections of the placenta encompassing the labyrinth zone, the junctional zone, and the decidua, and coronal sections of the brain, were analyzed for each animal. Cells were counted by an investigator blinded to the experimental conditions, and cell density was reported as the number of cells per objective field.

2.5. Cytokine Array

Cytokines were quantified in the placenta and amniotic fluid by the Mouse Cytokine Antibody Array kit. The placenta (E15.5) from each fetus was dissected from the uterus and pooled to represent the mean value for that particular litter (n = 1 per experimental group). The placental tissue was homogenized in cell lysis buffer (ThermoFisher Scientific, Waltham, MA, USA) and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected, and the total protein concentration was determined by a BCA kit (ThermoFisher Scientific, Waltham, MA, USA). The amniotic fluid (~100 μL) was drawn from each amniotic sac using an 18-G syringe needle and was combined to represent that particular litter. The cytokine array was performed according to the manufacturer’s instructions. Chemiluminescence signals were acquired by the ChemiDoc MP imaging system (Bio-Rad Laboratories, CA, USA). A vendor-supplied Excel-based software (version 4.1) was used to analyze the array data. The optical density of the target cytokines was normalized to positive controls. A threshold of a 1.5-fold change (increase or decrease relative to controls) was considered significant.

2.6. Western Blot

The placenta or forebrain was dissected and homogenized to prepare whole-cell lysates. Tissue was homogenized by sonication in ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na_4_P_2_O_7_, 2 mM Na_3_VO_4_, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, and protease inhibitors (MilliporeSigma, St. Louis, MO, USA). Lysates were clarified by centrifugation at 12,000× g for 10 min at 4 °C. The supernatants were collected, and the total protein concentration was determined. The samples were denatured and separated by SDS-PAGE on Bio-Rad TGX stain-free gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) using a semi-dry transfer system. Membranes were blocked with 5% non-fat dry milk in TPBS (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4) for 1 h at RT and incubated overnight at 4 °C with the NGF primary antibody diluted in blocking buffer (1:800). Following three washes in TPBS, membranes were incubated with HRP-conjugated secondary antibodies (1:20,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at RT. Chemiluminescent signals were detected using Clarity Max ECL substrates and acquired by the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Data were analyzed by the ImageLab software (version 6.1, Bio-Rad Laboratories, Hercules, CA, USA). The optical density (OD) of the target protein bands was normalized to the total protein load, as determined from stain-free gel images captured before chemiluminescent development.

2.7. Data Analysis

Data were analyzed using an unpaired t-test in SigmaPlot software (version 11. Systat Software Inc., San Jose, CA, USA). For postnatal datasets, a two-way ANOVA was first conducted to assess potential sex effects; as no significant sex differences were detected, male and female data were subsequently pooled within each experimental group for t-test analysis. For all statistical analyses, the litter was considered the experimental unit to ensure biological independence of all replicates. The final data are reported as Mean ± SEM, and p < 0.05 was considered statistically significant.

3. Results

3.1. MIA Leads to a Robust Inflammatory Response in the Placenta

Poly (I:C) is a synthetic double-stranded RNA that activates toll-like receptor 3 (TLR3), a pattern-recognition receptor highly expressed by innate immune cells. To determine the inflammatory profiles of the placenta after maternal poly (I:C) exposure, we first examined macrophages and neutrophils using their specific markers. As shown in Figure 2A, scattered F4/80+ macrophages were primarily detected in the decidua and labyrinth of control mice, while a strong infiltration of F4/80+ macrophages was observed throughout the placenta of poly (I:C)-treated mice (Figure 2B,I). The significantly enlarged cell bodies, along with intense immunofluorescence signals, indicate that these macrophages were highly active (Figure 2D). To gain further insight into their functional state, we performed immunofluorescence staining for CD68 (also known as ED1), a marker highly expressed by macrophages with enhanced phagocytic activity. CD68+ cells were rarely observed in the controls (Figure 2E) but were markedly increased in the poly (I:C)-treated mice (Figure 2F,J), consistent with their role as first responders to infection or inflammatory stimuli. Similarly, Ly6G+ neutrophils were absent in the controls but were abundant in the placenta of poly (I:C)-treated mice [Control: 0 ± 0, poly (I:C): 44.2 ± 7.9 cells/field, Figure 2G,H]. Together, these findings suggest that MIA led to significant infiltration of macrophages and neutrophils in the placenta from the maternal immune system.

Several studies have demonstrated that maternal and/or placental production of IL-6 and activation of its downstream IL-17A signaling pathway are key mediators of ASD-like behaviors in offspring following MIA. As an immunologically active organ housing diverse immune cell types, the placenta likely generates a broad spectrum of inflammatory mediators in response to maternal poly (I:C) exposure. To characterize the inflammatory profiles at the maternal-fetal interface, we compared levels of 96 cytokines associated with immune activation in the placenta tissue and amniotic fluid. Considering the semi-quantitative nature of the protein array, only cytokines with more than 1.5-fold change relative to controls were considered significant. Among 96 cytokines examined, 16 were increased and 2 were decreased in the placenta, whereas 30 were increased and none were decreased in the amniotic fluid. As shown in Table 2, the upregulated cytokines in the placenta can be grouped broadly into three categories: 1. Non-classic inflammatory cytokines associated with immune activation (Axl, CD27, CD27L, CD30, CD30L, CD36, CTLA-4, and sTNFR1); 2. Classic inflammatory cytokines (IL17A, IL1α, IL1β, and IL1ra) and chemokines (CXCL16, CCL5, CXCL13); and 3. Growth factor receptor (Amphiregulin). Two growth factor-related proteins (VEGFR, Osteopontin) were found to be downregulated. The cytokines upregulated in the amniotic fluid (Table 3) can also be grouped into 5 categories based on their functions: 1. proinflammatory cytokines (IL1β, IL2, IL3, IL6, IL12, IL15, IL17A, IL17E, IL17F, IL21), 2. anti-inflammatory cytokines (IL1ra, IL4, IL-5, IL10, IL13), 3. chemokines (CXCL1, CXCL9, CXCL11, CXCL15, CCL22, MCP1, MIP2, and MIP3α), 4. adhesion molecules (JAM-A, L-selectin, and CAM1), 5. Hormones and regulatory factors (leptin, leptin R, and MFG-E8, which is a secreted glycoprotein from immune cells that acts like an opsonin and plays a role in phagocytosis). It is worth noting that the cytokine landscape of the amniotic fluid differs from that of the placenta. A significantly more upregulated cytokines were observed in the amniotic fluid than in the placenta (30 vs. 16). In addition, the elevated cytokines in the amniotic fluid are more functionally diverse, notably many belong to the interleukin family (particularly IL6 and IL17A). This highlights a broader and more robust inflammatory signature in the amniotic fluid than in the placenta.

3.2. MIA Reduces Placental NGF Expression

The placenta produces several neurotrophic factors, including nerve growth factor (NGF), which supports neuronal survival, differentiation, and target innervation during early brain development [15]. To determine whether inflammation alters placental endocrine function, NGF expression was quantified by Western blot analysis. As shown in Figure 3, NGF was abundantly expressed in control placentas but was markedly reduced by maternal poly (I:C) administration.

3.3. MIA Activates Microglia in the Mouse Offspring

Microglial activation is frequently implicated in neurodevelopmental disorders such as ASD. Having established robust activation of the innate immune system at the maternal–fetal interface, we next examined microglial responses in P1 and P6 mouse offspring. As shown in Figure 4, MIA resulted in a marked increase in microglia, with the SVZ and hippocampus showing the most pronounced effects. Additionally, alterations in morphological characteristics are also notable between the MIA and control groups. Microglia of the MIA mouse offspring displayed enlarged somata with short, thick, and bushy processes, as compared to generally small somata with fine or minimal processes of the control mice. Some hypertrophic microglia contained multiple nuclei, likely reflecting phagocytic inclusions. Although these morphological differences were less pronounced at P6, microglial density remained substantially elevated compared with controls.

3.4. MIA Enhances NPC Proliferation in the Neurogenic Region

We previously reported that maternal LPS exposure markedly increases NPCs in the SVZ of P1 mouse offspring [13]. Because both LPS- and poly (I:C)-induced MIA paradigms generate ASD-like behavioral phenotypes, we next asked whether a similar alteration occurs in our poly (I:C)-based MIA model. We quantified Ki67^+^ NPCs in the SVZ and DG, two principal neurogenic niches of the early postnatal brain. Poly (I:C)-exposed pups exhibited a significant increase in Ki67^+^ cells in both regions at P1 (Figure 5A–C). To test whether an increase in NPC proliferation leads to enhanced neurogenesis, we quantified the expression of Dcx in the forebrain by Western blot. Unexpectedly, Dcx was greatly reduced by MIA (Figure 5D,E).

3.5. MIA Disrupts Cortical Neurogenesis

Disruption in cell cycle regulation of NPCs likely affects cortical development, given that cortical neurons are generated from NPCs within the SVZ in successive waves. To test this possibility, we quantified the proportions of early-, mid-, and late-born cortical neurons in the frontal cortex of P1 and P6 offspring mice, using their specific markers Tbr1, Ctip2, and Satb2, respectively. Quantitative analysis of cell counting revealed that the number of early-born Tbr1^+^ neurons in layer 6 of the cerebral cortex was significantly increased (Figure 6A,B,E). In contrast, the number of mid-born Ctip2^+^ neurons in layer V–VI was unchanged (Figure 6F). By P6, the later-born Satb2+ neurons in layers II–IV were also significantly increased in poly (I:C)-exposed mouse offspring (Figure 6C,D,G). These data show that MIA disrupts the temporal dynamics of cortical neurogenesis.

4. Discussion

Here, we report that maternal immune activation (MIA) elicited a strong innate immune response in the placenta, resulting in the release of a broad spectrum of inflammatory mediators, particularly Th17 cytokines, which have been implicated as key drivers of ASD-associated neuropathology and behaviors in mice. Notably, this Th17-biased inflammatory signature emerged alongside disrupted placental endocrine function and sustained microglial activation in the offspring, leading to impaired cell-cycle regulation and altered cortical neural progenitor dynamics during early brain development.

The association between placental inflammation and neurodevelopmental disorders has been reported in numerous clinical studies. For example, a case–control study involving 254 participants found that acute placental inflammation was associated with a significantly increased risk of ASD [16], while placental inflammatory cytokine mRNA expression was associated with poor cognitive performance in school-age children [17]. Preterm infants born to mothers with a history of placental histological chorioamnionitis were at high risk of neurodevelopmental disabilities [18]. The MIA animal model provides a valuable framework for elucidating the underlying mechanisms by which the placental inflammatory signals are transmitted to the fetal brain and how these pathways subsequently disrupt neurogenesis and neural circuit formation. Although notable inconsistencies exist among studies, converging evidence indicates that IL-6 and its downstream IL-17/RORγt signaling cascade play central roles in relaying maternal inflammatory cues to the developing fetal brain. For example, maternal systemic administration of IL-6 is sufficient to reproduce ASD-like behavioral abnormalities induced by poly (I:C) in mice, whereas IL-6 knockout or treatment with an IL-6-neutralizing antibody prevents these deficits [19]. Subsequently, it was demonstrated that maternally derived IL-6 activates JAK/STAT3 signaling within spongiotrophoblast cells of the placenta, an effect associated with reduced expression of placenta-specific hormones such as IGF-1 and growth hormone (GH) [20]. More recent work further demonstrated that Th17 cytokines, particularly IL17A, and maternal RORγt-expressing proinflammatory T cells are required for developing ASD-like behavioral phenotypes in poly (I:C)-exposed offspring mice [5,21]. Consistent with these reports, we observed a significant increase in IL-17A levels in both the placenta and amniotic fluid following MIA. It is worth noting that elevation of IL-6 was observed only in the amniotic fluid but not in the placenta, a pattern likely attributable to the timing of sample collection (96 h post-MIA). In an earlier study, we found that LPS-induced IL-6 production peaks at approximately 24 h but becomes undetectable by 96 h [22], suggesting that placental IL-6 elevation is transient. In addition to IL-6 and IL-17A, two key inflammatory cytokines linked to abnormal brain development in MIA models, a broader array of cytokines and other endocrine factors was detected in the amniotic fluid compared with the placenta. The sustained presence of these cytokines and hormones likely reflects additional contributions from other sources, including the amniotic and chorionic membranes, the fetal immune system, and/or maternal tissues.

Our data also indicate that placental endocrine function is impaired following MIA. We observed a seven-fold increase in leptin levels in the amniotic fluid. Leptin is not only a metabolic hormone but also a key placental endocrine factor produced by trophoblasts [23]. Increased placental leptin is associated with heightened inflammatory cytokines and oxidative stress in preeclampsia [24], whereas increased leptin levels in the amniotic fluid of pregnant women who later developed eclampsia were associated with a high risk of fetal intrauterine growth restriction (IUGR) [25], suggesting that the increase in leptin by the stressed and/or inflamed placenta reflects its endocrine dysregulation. Of note, elevated leptin levels in the amniotic fluid may also reflect a fetal contribution, as the fetus itself produces leptin [26]. In addition, the cytokine array analysis revealed that VEGFR was reduced in the placenta. This is a potentially important indicator of impaired endocrine dysfunction, given that VEGFR plays a key role in the formation and maintenance of the syncytiotrophoblast, the primary hormone-producing and nutrient-exchange layer of the placenta [27]. To validate this, we analyzed NGF expression levels in the placenta. NGF is known to be synthesized by the placenta to serve as a brain patterning molecule before robust neurogenesis begins. In agreement with the early report that the human placenta expresses NGF mRNA and protein [28], we detected abundant NGF proteins in the placenta of control mice. Critically, MIA markedly reduced placental NGF levels at embryonic day 15.5, a critical window of forebrain patterning and early neurogenesis. Although these findings support a potential link between placental endocrine disruption and altered neurodevelopment, we note that direct evidence demonstrating placental NGF transport across the fetal blood–brain barrier is currently lacking.

The cerebral cortex is essential for higher cognitive and emotional functions. Cortical development follows an “inside-out” sequence in which early-born neurons populate the deep layers (V–VI), whereas later-born neurons migrate away toward the pia surface to form the upper layers (II–IV). This laminar architecture emerges progressively, with neurons generated at different developmental time points acquiring distinct molecular and transcriptional identities. To assess the impact of MIA on cortical development, we quantified immature neurons in different cortical layers that reflect different timings of birth. Tbr1 and Ctip2 are expressed in deep-layer cortical neurons beginning in late gestation, whereas Satb2 expression initiates slightly later in upper-layer neurons and increases postnatally [17,29]. All three markers are readily detectable at P1 and P6, which span a critical window when cortical laminar identity is established but remains developmentally plastic [30,31]. Tbr1 is one of the high-confidence ASD genes (SFARI category 1) associated with language and cognitive impairment [32]. The finding that the number of Tbr1+ neurons was increased in MIA mice suggests that the temporal dynamics of cortical neurogenesis are disrupted. Because Tbr1 is a transcription factor expressed by early-born neurons in the deep-layer (layer VI) and plays a critical role in neuronal identity, migration, and connectivity [33], excessive generation of early-born Tbr1^+^ neurons may reflect prolongation or dysregulation of the neurogenesis window. This abnormal laminar patterning likely disrupts corticothalamic projections, influencing sensory processing and thalamic feedback loops—features that are associated with neurodevelopmental conditions linked to ASD and intellectual disability.

The number of late-born Satb2^+^ neurons was also increased in cortical layers II–IV of MIA mice at P6. Considering early-born Tbr1^+^ neurons were similarly increased, this may suggest a global acceleration of neurogenesis following MIA. However, this interpretation seems unlikely, given that the density of mid-born Ctip2^+^ neurons was unchanged. Moreover, expression of the neurogenic marker DCX was reduced in MIA mice, further arguing against this idea. The increase in Ki67^+^ NPCs in the SVZ and DG following MIA aligns with our previous findings in an LPS-based MIA model [13], indicating that NPC expansion in the neurogenic region is a shared feature of two classic MIA paradigms. Importantly, the literature suggests that MIA influences progenitor behavior through cell-intrinsic mechanisms. For example, NPCs isolated from poly (I:C)-exposed mouse offspring mice exhibit enhanced self-renewal capacity ex vivo [11]. Alternatively, the increase in NPC proliferation may be driven by factors associated with elevated microglial activity, as we previously observed a mixed M1/M2-like microglial phenotype in an LPS-based MIA model [13]. Collectively, these data suggest that MIA alters intrinsic cell-cycle regulation within NPCs, rather than simply enhancing endogenous mitogenic cues. However, whether excessive production of cortical immature neurons is a result of NPC expansion or disruption in temporal dynamics remains an open question that will be investigated in future studies.

There are several limitations of this study. First, due to the relatively small sample size, we were unable to reliably assess sex differences; therefore, all statistical analyses were performed using combined male and female data within each group. Second, behavioral assessments were not performed, precluding direct correlations between molecular changes and functional outcomes. Third, while our study provides evidence supporting an association between placental inflammation, endocrine dysfunction, and brain development, causal relationships remain unaddressed because no interventional experiments were conducted. In addition, cytokines and endocrine factors were measured at the protein level in placental tissue, which may not exclusively reflect placental origin and could include contributions from maternal or fetal sources. These mechanistic questions will be addressed in future studies. Nevertheless, our findings reinforce the emerging concept that the placenta plays an active role in programming fetal brain development, and that disruption of placental homeostasis is closely associated with an increased risk of neurodevelopmental disorders such as ASD.

5. Conclusions

Our findings demonstrate that MIA triggers a placental Th-17-biased inflammatory response, disrupts placental immune and endocrine homeostasis, and profoundly reshapes fetal brain development. The convergence of heightened placental inflammation, impaired trophoblast-derived hormonal support, sustained microglial activation, and altered neural progenitor dynamics provides a mechanistic framework linking maternal inflammation to aberrant cortical patterning. These results reinforce the concept that placental pathology plays a key role in driving ASD-like neuropathology and behaviors in the offspring.