Transient episodes of EIA can trigger persistent behavioral and molecular adaptations in mice. It is important to emphasize that many of these changes—particularly the molecular changes—were evident long after the acute immune response was triggered. With respect to behavioral endpoints, MIA (Poly I:C administered on E12.5) tended to have minimal effects, whereas PIA (LPS administered on PND9) produced more pronounced effects. In most—though not all—cases, the most dramatic effects were seen with the “two-hit” (combined) treatment (prenatal Poly I:C plus postnatal LPS). Likewise, there were sex differences across some—though not all—behavioral domains, with males being more reliably and robustly affected. With respect to molecular endpoints, we examined changes in mRNA and protein levels for inflammation-related genes (e.g., pro-inflammatory, neuroinflammation, and anti-inflammatory markers) in mice that had received behavioral screening and found both qualitative and quantitative differences. The most striking finding was evidence for fundamental sex-related differences in the type of immune response triggered by perinatal immune activation. We found that both males and females showed significant increases in the expression of pro-inflammatory and neuroinflammation markers in the brain, and that the changes tended to be larger in male mice across all areas examined (mPFC, AMG, HP, and TM). However, we discovered that males and females showed opposite patterns of changes in anti-inflammatory markers: specifically, both IL-10 and TGF-β1 were down-regulated by perinatal immune activation in males, whereas these same markers were significantly up-regulated in females. Our findings suggest that immune system activation during critical periods of development is sufficient to produce persistent alterations in behavior and brain biology—even in the absence of accompanying genetic vulnerability or injury—and that females may mount anti-inflammatory responses that may explain reduced severity of the phenotype in mice of this sex.

Behavioral changes

MIA on its own produced few effects on behaviors that resemble the core features of ASD in either sex. This outcome was surprising because some of the seminal reports on EIA13,14 described profound ASD-like phenotypes in tests quantifying social behavior, ultrasonic vocalizations, and repetitive behaviors. We have also shown negligible effects of prenatal Poly I:C alone on behaviors such as time in the center of an open field17, and sleep and epileptiform activity11. Recent work suggests that gut bacterial flora, which can differ among commercial suppliers and institutional animal care facilities, plays a critical role in the ability of prenatal Poly I:C to produce behavioral effects. Indeed, we purchased our mice from a supplier (Jackson Labs) that lack a commensal segmented filamentous bacteria in the gut that cause induction of an immune factor (IL-17a) that regulates sensitivity to the Poly I:C18,32, providing a potential explanation for our lack of strong effects. Alternatively, the lack of reliable Poly I:C effects could also be related to batch variability; indeed, we used multiple batches of Poly I:C that might conceivably have differences in potency or efficacy32. In contrast, PIA tended to be sufficient to produce signs of the 3 core features of ASD. Whereas communication-like behavior was affected in both sexes—with virtually identical effects on the numbers, patterns, and durations of USVs—changes in social behavior in a social approach test25 were observed only in males, with no effects whatsoever in females. In addition, LPS treatment produced strong effects in the social scent test, which has been validated only in males24. Male mice treated with LPS only also showed enhanced performance on the rotarod; while this test is often used to quantify motor performance capabilities, there is precedent in the literature for interpreting it as an indicator of repetitive or restricted movements31. The behavioral effects were generally strongest with the two-hit combination, although in the case of rotarod behavior, the early Poly I:C effect appeared to have a protective-like effect that was unexpected and is difficult to explain. Finally, males showed larger increases in anxiety-like behavior—which is often a characteristic of ASD in humans33 despite not being a diagnostic criterion for the condition—in males but not females, without accompanying non-specific alterations in locomotor activity that might complicate data interpretation. These patterns of behavioral changes, together with their similar ability to produce alterations in sleep and epileptiform behavior11—which are frequently co-morbid with ASD—provide support for this model in the context of ASD research. In addition, the fact that we found in the behavioral tests that males are more frequently affected than females is consistent with the higher prevalence of ASD in males (4:1), and suggests sex-dependent differences in the distribution of traits regulating behavior.

Molecular changes

EIA produced elevations in pro-inflammatory (TNFα, iNOS, IL-6, and IL-1β) and neuroinflammation markers (Iba-1, GFAP, and TSPO). Effects were observed in both sexes; while the effects tended to be larger in males than in females, these studies were intended to yield a qualitative “first pass” analysis of the existence of potential sex differences on a large number of markers rather than a rigorous quantitative comparison of a more restricted list. Qualitatively similar effects were seen with mRNA and protein, although increases in mRNA levels in males were on occasion considerably higher than those seen in females despite the similarities in levels of protein, raising the possibility of post-translational effects that limit the scale of changes in protein expression. Such drastic increases in innate immune mRNA, with relatively smaller increases in protein levels parallel similar findings in microglia that are the result of translational suppression mechanisms34. There was a small number of specific cases where increases in mRNA levels of pro-inflammatory/neuroinflammation markers were not accompanied by changes in protein expression, also suggesting post-translational processes that further regulate protein expression. These individual cases in specific brain regions do not detract from the significance of the often massive changes in both mRNA and protein levels in other brain areas. Our findings in brain are broadly consistent with the existing literature. Perinatal immune activation in mice also increases TNFα, IL-6 and IL-1β mRNA levels in placenta35,36, and single maternal injection of IL-6 resulted in ASD-like signs in the offspring13. Interestingly, male mouse pup-derived astrocyte cultures show markedly higher expression of TNFα, IL-6 and IL-1β mRNA in response to LPS compared to those derived from females37. Collectively, the present studies are consistent with data from humans. Altered inflammatory cytokine levels in postmortem brain samples and serum plasma samples from individuals with ASDs have been previously reported in numerous studies. For example, pro-inflammatory cytokines such as TNFα, IL-6 and IL-1β are reportedly elevated38,39,40. In addition, TLR3 and TLR4 activation upregulates cytokine expression in peripheral blood monocyte cultures from children with ASD41, although sex differences were not examined. Similarly, while sex differences were not examined, neuroinflammation markers are also elevated in individuals with ASD42,43,44,45,46,47,48.

Although we found the direction of changes of pro-inflammatory markers were qualitatively similar in male and female mice, there were major sex differences for anti-inflammatory markers. Perinatal immune activation produced decreases in mRNA and protein levels of IL-10 and TGF-β1 in males, but increases in the same markers in females. There is little evidence in the literature that such differences have been previously studied or described. LPS produces decreases in IL-10 levels in macrophage cultures from BTBR T + tf/J (BTBR) mice, which show ASD-like features49. Interestingly, female rats had significantly higher levels of mRNA for IL-10 and its receptor IL-10ra than males in the cortex and HP during normal development (at P0, P4 and P60)50. Anti-inflammatory cytokines such as IL-10 and TGF-β1 are decreased in serum from individuals with ASD51,52,53, although sex differences were not examined. The molecular mechanisms underlying sex-different gene regulation by perinatal immune activations are currently unknown, but it is likely that genes linked to sex chromosomes, hormonal changes, and/or their interactions may underlie these differences54. The fact that Poly I:C and/or LPS both produced changes in these markers, whereas Poly I:C consistently produced negligible effects on behavior, suggests that molecular techniques have a sufficiently high sensitivity to detect effects that are below the threshold needed to significantly alter behavior, as was seen previously in electrophysiology studies17. Regardless, our findings of sex differences in anti-inflammatory responses raise the possibility of protective (resilience-related) processes that reduce the prevalence of ASD in females.

Limitations of the paradigms

Our studies characterized the effects of using Poly I:C as the trigger for MIA and LPS as the trigger for PIA. We used this treatment order because of evidence showing that TLR3 expression is high but steadily decreasing during prenatal development, whereas TLR4 expression is low but steadily increasing over the same period55. Postnatal LPS administration also models the high risk of exposure to bacterial infections occurring during this period of development. PND9 in a mouse approximates neurodevelopmental milestones—including brain growth, gliogenesis, and increases in axonal and dendritic density—present in humans at or slightly before, full-term birth56. In humans, labor produces over a 10-fold increase in microbial invasion of the amniotic cavity (MIAC)57,58, and pre-term birth is highly associated with MIAC and is a risk factor for ASD58,59. Furthermore, neonates are at high risk for bacterial infections60. Other permutations of this treatment regimen, or experiments in which gene X environment interactions are examined, may yield different outcomes. There are several important caveats with our measurements of immune markers in the brain. Because rapid dissection was used, we cannot exclude peripheral blood as an important source for the cytokines detected here. However, changes in astrocyte and microglia markers support inflammatory changes in the central nervous system in this model. Additionally, many of these cytokine markers are normally found at low levels in the brain, which while allowing accurate detection of qualitative elevations from baseline can lead to imprecision in the measurement of baseline levels or the magnitude of change.

Implications

These findings provide support for the utility of MIA, PIA, and combined treatment for the study of ASDs on several levels. First, the patterns of behavioral changes, together with their prevalence in males, fit well with the condition as it occurs in humans. The observation that both males and females mount persistent pro-inflammatory responses, whereas only females mount accompanying anti-inflammatory responses, represents a new and potentially important discovery that might aid in understanding sex differences in prevalence and tailoring new treatments. Together, these findings add to accumulating evidence for an immune subtype of ASD7,61, and suggest that immune-related pathophysiologies can differ between sexes. The ability to better identify subtypes of this syndrome will facilitate care and perhaps provide a justification for investigations into treatment strategies (e.g., immune-modulating agents) that might not otherwise be considered for ASDs.