22 apr. 2025
Ellul et al. (BioRixv)
Keywords
Maternal immune activation (MIA)
T regulatory cells (Tregs)
T helper 17 cells (Th17)
Autism
Main Findings
In this preprint, the authors investigate the role of maternal immune activation (MIA) in the development of autism spectrum disorder (ASD), hypothesizing that disruptions in maternal immune responses during pregnancy can increase the risk of ASD in offspring through mechanisms involving regulatory T cell (Treg) dysfunction. Building on prior research linking interleukin (IL)-17 signaling in utero to ASD development and noting that autistic individuals show reduced Treg populations and elevated IL-17A levels in serum, the authors focus on understanding how Treg dysfunction influences both immune and neurodevelopmental outcomes in maternal and offspring contexts. To address these questions, they employ a variety of techniques, including epigenetic analysis, bulk and single-cell RNA sequencing (scRNA-seq), functional assays, and histological and novel imaging analyses to probe the effects of MIA on Treg function. They demonstrate that MIA results in a significant reduction in maternal Treg populations, and importantly, pre-emptive administration of low-dose IL-2 (IL-2LD) prior to MIA induction rescues ASD-like behaviors in offspring. These behaviors include impaired communication, increased repetitive behaviors, and reduced social interaction. Beyond the behavioral improvements, the study reveals that MIA leads to lasting epigenetic modifications, notably the downregulation of vitamin D receptor (Vdr) and upregulation of brain enriched myelin associated protein 1 (Bcas1), both of which are implicated in ASD and neurodevelopmental processes. To further substantiate the functional deficits in offspring Tregs, the authors assess the diabetes susceptibility in non-obese diabetic (NOD) mice, which are inherently susceptible to spontaneous diabetes due to Treg defects. They demonstrate that MIA-exposed offspring from NOD mice develop diabetes more rapidly and at an earlier age than their controls, underscoring the Treg dysfunction induced by MIA. Moving beyond phenotypic and suppressive characterizations, they then provide further evidence using scRNA-seq to reveal chronic meningeal inflammation in MIA-F1 offspring. This inflammation is marked by increased monocyte and neutrophil populations, a pro-inflammatory transcriptome, and disruption of neurodevelopmental pathways. Notably, these pathological changes were reversed by IL-2LD treatment, indicating the potential for Treg modulation to mitigate the immune and neurodevelopmental impairments induced by MIA. Finally, the authors demonstrate that postnatal intervention with Adeno-associated virus (AAV)-IL-2, a vector designed to provide long-term Treg stimulation, rescues ASD-like behavioral deficits in adult MIA-F1 mice. This intervention alleviates social impairments and repetitive behaviors by stimulating Tregs and reducing both brain and meningeal inflammation, reinforcing the central role of Tregs in neurodevelopment. This study provides compelling evidence that maternal Treg dysfunction is a key contributor to the development of ASD-like behaviors in offspring. Importantly, the study highlights the potential of IL-2-mediated Treg stimulation for neurodevelopmental disorders linked to immune dysfunction, offering promising insights for future clinical interventions.
Limitations
While the authors justify the use of the Poly I:C (PolyIC) model of maternal immune activation (MIA) as an effective method for inducing IL-17-mediated autism spectrum disorder (ASD) phenotypes in offspring—and this is further supported by others in the field—it raises the question of whether additional models could have bolstered their findings. For instance, alongside the PolyIC model, the authors could have incorporated the PRIMA-17 model (Andruszewski et al., Mol Psychiatry,2024), which enables the transgenic transfer of IL-17A across the placental barrier, inducing behavioral deficits in offspring. This complementary approach may have strengthened the translational and mechanistic impact of their study.
To examine the impact of prior Treg depletion on MIA-induced outcomes, the authors utilized FOXP3-DTR mice to ablate FOXP3⁺cells in mothers. However, this prenatal depletion approach resulted in a 90% abortion rate, significantly limiting the interpretability and reproducibility of the findings. From the surviving 10%, the authors report severe behavioral impairments, surpassing those seen with MIA alone. These results, while compelling, would have been more robust if supported by additional strategies, such as partial depletion models to preserve fetal viability or conditional knockout systems targeting key windows of postnatal neurodevelopment.
The authors propose that epigenetic reprogramming underlies Treg dysfunction in MIA-exposed offspring, supported by differential expression of genes such as Vdr and Bcas1. However, the manuscript would benefit from follow-up functional assays to validate the altered expression of Vdr and Bcas1in Tregs of MIA-F1. Functional characterization—such as in vitro Treg polarization under Th17-skewing conditions, or metabolic profiling—could have enhanced the mechanistic insights.
A classical in vitro suppression assay was employed to evaluate splenic Treg function, with no observed differences between groups. While this result is informative, and in agreement with the authors, such polyclonal suppression assays are often limited by their dependence on IL-2 availability and do not fully capture context-specific Treg functionality. Additional experiments – for example the adoptive transfer of Tregs into MIAF1 offspring, or more detailed characterization of Tregs in the offsprings’ tissues via flow cytometric profiling of suppressive markers in their experimental groups (e.g., FOXP3, CTLA-4, CD25, GITR, Neuropilin-1, CD39/CD73) - would support the authors’ findings and provided a more comprehensive picture of the proposed Treg deregulation in MIAF1 animals. It would be very interesting to pair such observations with a characterization of maternal Tregs.
The use of AAV vectors to deliver IL-2 for long-term Treg stimulation presents a promising therapeutic approach; however, the manuscript would benefit from an assessment of Treg exhaustion under these conditions. Chronic IL-2 exposure can lead to altered Treg stability or function, and this should be carefully monitored in future studies using phenotypic markers, exhaustion signatures, or longitudinal assessments of Treg fitness.
The authors employed several field-standard behavioral assays to differentiate ASD-like phenotypes from typical behavior. However, the study does not clearly distinguish whether the IL-2 treatment was assessed for its preventative or therapeutic potential in the offspring. There is no indication of whether treatment was administered before or after the onset of ASD-like symptoms. A time-course or kinetic analysis outlining when behavioral abnormalities first appear in MIA offspring would have strengthened the interpretation of treatment effects. Based on the methods, IL-2 was administered around postnatal day 21, but it remains unclear whether ASD-like behaviors had already emerged at that point or whether this timing served as a true post-symptom intervention. Furthermore, it remains unaddressed whether these ASD-like behaviors persist beyond early postnatal life or whether they diminish over time as inflammation resolves. It would be important to assess whether MIA offspring still exhibit ASD-like behaviors if tested at later developmental stages (e.g., 3–4 months of age). This would help determine whether the observed phenotype is transient or stable over time. Finally, while ASD is understood clinically as a spectrum disorder, the behavioral phenotyping here is treated in binary terms (ASD-like vs. not). A more nuanced approach—such as testing a range of IL-2 doses to evaluate whether increasing Treg stimulation correlates with varying degrees of symptom severity—could provide important insight into how immune modulation influences both the severity and spectrum of ASD phenotypes.
The authors utilized several behavioral assays to assess ASD-like phenotypes in the offspring; however, the focus was primarily on early postnatal to early adult stages. It would be valuable to examine the long-term effects of MIA in offspring to determine whether the early improvements observed with treatment are sustained into adulthood. Furthermore, in clinical settings, ASD in females is often not diagnosed until much later in life. This raises an important question: for individuals who miss early diagnosis and intervention, what therapeutic options remain? In vivostudies examining whether IL-2 treatment can still ameliorate ASD-like phenotypes when administered later in adulthood (e.g., 4–12 months of age in rodents) would offer important insight into the therapeutic window and potential for late-stage intervention.
ASD is widely recognized to have a strong male bias, with approximately four males affected for every one female. In the NOD experiment, however, the authors reported that female—but not male—offspring of MIA dams developed diabetes more rapidly and frequently. Interestingly, this was the only experiment in the study that addressed sex differences, and the findings ran counter to clinical observations. Further research is warranted to better understand the sex-specific effects of MIA-induced ASD and the differential responses to IL-2 treatment.
The use of both histological and imaging techniques to assess brain structural changes was well-executed. However, several studies report that individuals with ASD often exhibit varying degrees of myelination defects. Given that IL-2 therapies are currently being explored for neurodegenerative and demyelinating diseases, it would have been valuable to also assess the state of myelination. It would have been particularly compelling if clinical observations were mirrored in the model—specifically, if MIA induced demyelination and AAV-IL-2 treatment promoted any degree of remyelination.
Significance/Novelty
This preprint represents a significant advancement in understanding the intersection of immunology and neurodevelopment, especially in the context of ASD The study’s novel contribution lies in demonstrating that MIA affects offspring behavior through Treg dysfunction, offering a new insight into how immune-mediated pathways may contribute to the development of neurodevelopmental disorders. Additionally, the research adds to the emerging field of central nervous system (CNS)/meningeal Tregs, highlighting their previously underexplored role in maintaining neuronal homeostasis. This finding opens new avenues for further investigation into how these Tregs contribute to brain function and neurodevelopment, broadening our understanding of neuroimmune connections.
For patients, the findings have profound implications for identifying novel therapeutic strategies to manage or prevent ASD linked to maternal immune activation. The use of low-dose IL-2, an immune-modulating treatment already in use for conditions such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and multiple sclerosis (MS), offers a potential avenue for therapeutic intervention. If low-dose IL-2 can be shown to mitigate ASD-like behaviors in at-risk children—particularly those exposed to maternal infections or immune dysfunction during pregnancy—this could pave the way for preventive or personalized treatments tailored to individual immune profiles. Such interventions could drastically alter the treatment landscape for ASD, particularly for those with an immune-mediated component. Additionally, the study highlights epigenetic alterations in Tregs as a crucial factor in ASD development. This finding offers an exciting prospect for developing epigenetic therapies or biomarker-based approaches that could help identify at-risk individuals early on, enabling timely interventions before full-blown neurodevelopmental disorders manifest.
Credit
Reviewed by Mahdieh Golzari as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.
The author declares no conflict of interests in relation to their involvement in the review.