Among the many well established environmental factors that can impact fetal neurodevelopment, asthma and air pollution represent two major sources of immune stimulation
that are on the rise, making them a significant concern for pregnant individuals. Based on previous studies of maternal immune activation with asthma, and PM exposure during pregnancy, we hypothesized that the combination of these two environmental stimuli would result in an exacerbated neuroimmune response in offspring. Although the appearance of an additive or synergistic effect of MAA and UIS exposure combined was limited, we did identify increases in cytokine concentrations across all treatment groups in the cortex and hippocampus that may suggest converging pathways for each insult/exposure. Importantly, some of these elevations appear to be sustained across treatment groups from adolescence into early adulthood in the cortex, demonstrating lasting impacts of these gestational exposures on the neuroimmune environment later in life. Although we identified increases in cytokines in the hippocampus within all treatment groups at P15, these did not remain elevated into early adulthood. Overall, these data show that MAA and UIS environmental stimuli can result in an altered neuroimmune environment that persists from juvenile to adult timepoints.
The allergic response in the lungs of individuals with asthma is characterized by an influx of immune cells, such as neutrophils, mast cells, macrophages, and T-helper (TH)2 cells. Our mouse model of MAA previously showed elevated IL-4, IL-5, IL-17, and IFNγ in the lung and peripheral blood of mice exposed to aerosolized OVA during pregnancy (Schwartzer et al., 2017; Church et al., 2021; Tamayo et al., 2022). These increases in maternal serum cytokines result in neuro-immune signaling changes in the fetal brain during in utero development (Tamayo et al., 2022). Our present data extend these findings by revealing evidence of increases in cortical and hippocampal cytokines in juvenile mice of MAA dams. In the cortex, for example, MAA alone increased IL-1β, IL-2, IL-17, IL-1α, IL-10, and IL-17. In the hippocampus, we identified IFNγ, IL-1β, IL-2, IL-7, IL-13, IL-15, MIP-1β, and RANTES as being elevated in P15 offspring of MAA-exposed dams. These observed increases in MAA compared to PBS-AIR controls demonstrates the independent neurodevelopmental impact of allergic inflammation during pregnancy on offspring neuroinflammation. In addition to these findings in the MAA only treatment group, we also identified the UIS treatment (in the absence of MAA) resulting in an increase of cytokine concentration in juvenile offspring, specifically, in IL-1β, IL-2, IL-13, IL-17, IP-10, MIP-1α, and IL-10. Moreover, increases in cytokines as a result of UIS exposure alone were also identified in the hippocampus of juvenile offspring, with elevated IFNγ, IL-1β, IL-2, IL-7, IL-12(p40), IL-13, IL-17, and RANTES. To the authors’ knowledge, investigations into these neurobiological outcomes in offspring under UIS exposure during gestation have not been previously reported, highlighting the novelty of our model and findings.
In addition to the independent effects of MAA or UIS treatment on cytokine concentrations in the cortex and hippocampus of juvenile offspring, these elevations were most often coupled with elevations in the MAA-UIS combined treatment group. Most notably, we observed elevations in the cortex of IL-1β, IL-2, IL-13, and IL-17 in dual-exposed MAA-UIS offspring. These cytokines were also trending or increased in the MAA and UIS single treatment groups as well as the MAA-UIS treatment group at P15, and they remained elevated into the P35 timepoint. Our data suggest a sustained elevation in these four cytokines from P15 to P35 as a result of both MAA and UIS that have the potential for long-lasting impacts on neurodevelopment in the cortex of these offspring.
Consistent with the pleiotropic nature of cytokines in the central nervous system (CNS), IL-1β, IL-2, IL-13, and IL-17 have all been identified as having neurotrophic properties (Awatsuji et al., 1993; De Araujo et al., 2009; Park et al., 2018; Lock et al., 2002; Kebir et al., 2007; Milovanovic et al., 2020; Ma et al., 2014; Rochman et al., 2013; Brombacher et al., 2017). Indeed, high concentrations (500 ng/mL) of IL-1β can have neurotoxic effects on neurons when exposed for 3–5 days (Park et al., 2018), and IL-17 is detected at high levels in the CNS in multiple sclerosis and associated with the neuroinflammatory pathology of the disease (Lock et al., 2002; Kebir et al., 2007). Compared to these neurotoxic concentrations, our data represent moderate increases in cytokines with less than 2.5-fold increases in treatment groups compared to PBS-AIR controls, and may not represent overt inflammation per se. However, these smaller changes in brain cytokine levels during the juvenile period may be biologically significant given their alternative functions in promoting neuronal survival and neurodevelopment. For example, IL-1β acts as a chemokine that guides neurite outgrowth in cortical neurons (Ma et al., 2014), and IL-17 acts in initiating the release of brain-derived neurotrophic factor (BDNF), glia-derived neurotrophic factor (GDNF), and nerve growth factors (NGF) associated with neuronal cell survival and repair (Milovanovic et al., 2020). Taken from this view, these cytokines, which are generally considered overtly inflammatory, may be having a more subtle impact on the neuroarchitecture of offspring brains than models finding dramatic increases in concentration of IL-1β and IL-17.
Further demonstrating the neuropoietic nature of these cytokines within the CNS and adding to the idea that the moderate increases observed in this model may be altering the neuropatterning of the offspring brains, IL-2 has been found to have neurotrophic properties and is necessary for proper cytoarchitecture in development (Beck et al., 2005; Beck et al., 2005). In addition, IL-13 is often considered anti-inflammatory in the CNS, with some studies pointing to a neuroprotective impact in CNS diseases and injuries (Miao et al., 2020; Guglielmetti et al., 2016; Le Blon et al., 2016; Pan et al., 2013). Both IL-2 and IL-13 are among several cytokines that are known to decrease in concentration at P14 under homeostatic conditions, and this developmental timepoint in mice is characterized by a high degree of synaptogenesis and pruning (Garay et al., 2013; Morato Torres et al., 2020). In contrast, our model, which investigated cytokine concentrations at P15, still within this window of high synaptogenesis, showed increased IL-2 and IL-13, representing a shift in homeostatic load. Taken together, it may be that these sustained moderate increases in cytokines of the cortex are changing the trajectory of cortical development and promoting altered connectivity in the cortex linked to behavioral changes such as decreased social interaction and repetitive behaviors previously identified in our model (Schwartzer et al., 2015; Church et al., 2020). This phenomenon of altered connectivity has also been implicated in the core behaviors associated with ASD, specifically the social deficits and restrictive and repetitive behaviors (Conti et al., 2017; Maximo et al., 2014; Mills et al., 2016).
Similar to our findings in the cortex, we also identified elevations in several cytokines, most notably IFNγ and IL-7, at P15 in the hippocampus of MAA alone, in UIS alone, and MAA-UIS offspring compared to controls. IFNγ receptors are present on both neurons and glia (Ottum et al., 2015). In the hippocampus, IFNγ appears to play a role in synaptogenesis (Lee et al., 2006). Some investigators have found that overexpression resulted in increased neurogenesis in the dentate gyrus, and because of its neuromodulatory effects, it has been suggested that this may impact cognition and social behavior as a result (Flood et al., 2019; Filiano et al., 2016; Baron et al., 2008). Additionally, IL-7 promotes survival and neurite outgrowth in hippocampal neurons (Macia et al., 2010; Michaelson et al., 1996). Considering the effects of IL-7 and IFNγ, and that the hippocampus is a major neurogenic niche in the developing brain, future studies may benefit from investigating the potential for hippocampal overgrowth in offspring brains in response to UIS or allergic asthma inflammation during pregnancy. Indeed, this phenomenon of hippocampal overgrowth has been identified in cases of ASD (Groen et al., 2010; Murphy et al., 2012, Rojas et al., 2006) and has been implicated in the deficits associated with emotion perception and sensory processing in ASD individuals (Groen et al., 2010, Banker et al., 2021). Curiously, the observed increases in hippocampal cytokine concentrations at P15 were not observed in any treatment group of P35 offspring compared to PBS-AIR controls. Although we can only speculate about these findings, it may be that these changes resolve during adolescence when additional brain maturation may be present to compensate for developmental overgrowth much in the same way that volumetric increases in the hippocampus of ASD individuals do not persist into adulthood (Groen et al., 2010; Rojas et al., 2006). Although IL-7 and IFNγ are only two examples of cytokines that we found elevated among the treatment groups, they illustrate the broader findings that treatment with MAA, UIS, or MAA-UIS can alter the hippocampal neuroimmune environment with potential consequences to behavioral outcomes.
Prenatal insults have been shown to have lifelong impacts on microglia function and are suspected to play a prominent role in neurodevelopmental disorders (Vogel Ciernia et al., 2018; Eggen et al., 2013; Knuesel et al., 2014; Slusarczyk et al., 2015; Giovanoli et al., 2016; Hughes et al. 2023). In ASD, some postmortem studies have identified differences in microglia density and morphology in brains of individuals (Koyama et al., 2015; Morgan et al., 2010; Morgan et al., 2014). In our previous study of MAA, we found DNA methylation differences in adult microglia, and several of these changes occurred in regulatory genes that are shared among some ASD individuals (Vogel Ciernia et al., 2018). Given these findings, we sought to examine the density of microglia in the P15 brains of our MAA and UIS exposure model. We observed a significant increase in microglia density within the hippocampus of offspring exposed to MAA-UIS, but these increases were not present in the frontal cortex. One plausible explanation for why these increases were only observed in the hippocampus may be due to the higher density of microglia known to be present in the hippocampus. This higher density of microglia is thought to make the hippocampus more vulnerable to inflammation (Réus et al., 2015; Choi et al., 2011), and disruptions in the dentate gyrus have been linked to neurodevelopmental disorders (Cai et al., 2018; Yagishita et al., 2017). Our findings of increased microglia density in the hippocampus of MAA-UIS offspring mirror data from another maternal immune activation model that utilizes the viral mimic poly(I:C). Specifically, Juckel et al. (2011) reported an increase in microglia density in the hippocampus but not the cortex of offspring born from immune-activated dams. Similarly, another study of maternal immune activation using LPS stimulation showed an increase in microglia density in the hippocampus (Diz-Chaves et al., 2013). While it is difficult to make conclusions about microglial function based on density data alone, our observed difference in the hippocampus in combination with similar reports from other maternal immune activation models (Juckel et al., 2011; Diz-Chaves et al., 2013) suggest that asthma allergy and PM mediated immune activation during pregnancy can result in a deviation from homeostatic activity in the offspring hippocampus.
Although we did not collect maternal data in this preliminary study, data from previous MAA studies demonstrates increased systemic inflammation characteristic of an allergic asthma response, specifically with increased IL-4, IL-5, and IL-13 (Schwartzer et al., 2017; Church et al., 2020; Tamayo et al., 2022), suggesting the potential for a similar response in dams of MAA in the current model. Speculation about the systemic impacts of UIS on the maternal immune system, however, is difficult. Many studies of PM exposure suggest IL-6, IL-8, and TNFα as the main cytokines upregulated in response to PM exposure (Mitschik et al., 2008; Silbajoris et al., 2011; Musah et al., 2012; Gong et al., 2022; Longhin et al., 2018). This difference in cytokine response highlights the potential reason we see differences in the impact between MAA and UIS in our model. However, models of PM can vary widely in the size of PM and composition (Mitschik et al., 2008), making speculation about the maternal response in the UIS groups, and the potential role this plays in offspring neurodevelopment, difficult. This variation in PM studies underscores the need for future investigations to identify the maternal cytokine milieu in this model.
While our findings do not necessarily demonstrate an additive effect of MAA and UIS with regard to the cytokines we investigated, we did see a synergistic impact of MAA-UIS on microglia density in the hippocampus. These findings demonstrate the potential for additive effects of maternal asthma exposure when coupled with PM exposure. Independently, studies have shown in both humans and animal models that PM exposure during pregnancy can increase the susceptibility of offspring developing asthma (Mortimer et al., 2008; Wang et al., 2013; Hua et al., 2023). This increased susceptibility of asthma in offspring is also seen in children of asthmatic mothers (Martel et al., 2009; Mattes et al., 2013; Murphy 2022), suggesting the potential for systemic immune disfunction when these two stimuli are combined during pregnancy. The findings in this unique model of MAA and UIS exposure highlight the importance of investigating the impact of these closely linked and prevalent environmental factors.