Offspring behavioral outcomes following maternal allergic asthma in the IL-4-deficient mouse

Abstract

Maternal allergic asthma (MAA) during pregnancy has been associated with increased risk of neurodevelopmental disorders in humans, and rodent studies have demonstrated that inducing a T helper-2-mediated allergic response during pregnancy leads to an offspring behavioral phenotype characterized by decreased social interaction and increased stereotypies. The interleukin (IL)-4 cytokine is hypothesized to mediate the neurobehavioral impact of MAA on offspring. Utilizing IL-4 knockout mice, this study assessed whether MAA without IL-4 signaling would still impart behavioral deficits. C57 and IL-4 knockout female mice were sensitized to ovalbumin, exposed to repeated MAA inductions, and their offspring performed social, cognitive, and motor tasks. Only C57 offspring of MAA dams displayed social and cognitive deficits, while IL-4 knockout mice showed altered motor activity compared with C57 mice. These findings highlight a key role for IL-4 signaling in MAA-induced behavioral deficits and more broadly, in normal brain development.

Graphical Abstract

1.0. INTRODUCTION

Pregnancy marks a critical period for the fetus when environmental factors impact brain development and later risk for neurodevelopmental and neuropsychiatric disorders (Barker, 2004; Doi, Usui, & Shimada, 2022; Han, Patel, Jones, & Dale, 2021). Changes in the immune environment during pregnancy are commonly associated with neurodevelopmental disorders including Autism Spectrum Disorder (ASD) and Attention Deficit Hyperactive Disorder (ADHD) (Han, Patel, Jones, Nielsen, et al., 2021; Meyer, 2019). Epidemiological findings report an increased risk of ASD among children born from mothers with asthma episodes, allergies, and autoimmune diseases during pregnancy (Croen, Grether, Yoshida, Odouli, & Van de Water, 2005; Li, Tsai, Hsiao, Chen, & Yen, 2022), with more severe social behavior impairments correlated with maternal asthma (Patel et al., 2018). Similarly, maternal asthma, autoimmune, and atopic diseases are associated with increased risk of ADHD in children (Cowell, Bellinger, Wright, & Wright, 2019; Nielsen, Benros, & Dalsgaard, 2017). Given the prevalence of allergic asthma as a chronic disease (Pawankar, Canonica, Holgate, & Lockey, 2012; Simpson et al., 2001) and the widespread presence of allergic triggers, including cockroach and mouse allergens, pollutants, and psychosocial stress (Matsui et al., 2008; Togias, Fenton, Gergen, Rotrosen, & Fauci, 2010), there is a pressing need to mechanistically understand the link between allergic asthma during pregnancy and altered offspring neurodevelopment.

Several models in rats (Breach, Dye, Galan, & Lenz, 2022; Breach et al., 2021) and mice (Church, Tamayo, Ashwood, & Schwartzer, 2021; Schwartzer, Careaga, Chang, Onore, & Ashwood, 2015) utilize ovalbumin (OVA) hypersensitivity to induce airway hyperresponsiveness and subsequent allergic asthma inflammation to test the impact of maternal allergic asthma (MAA) on offspring behavior. In mice, MAA elevates peripheral cytokines, most notably interleukin (IL)-4 and IL-5 (Church et al., 2021; Schwartzer et al., 2017), closely mirroring cytokine profiles reported in humans following episodes of allergic asthma. Offspring in these rodent models show strain-dependent alterations in social interaction, increased repetitive digging behavior, and sex-specific changes in anxiety-associated responses (Church et al., 2021; Schwartzer et al., 2015; Schwartzer et al., 2017), accompanied by region-specific changes in brain cytokine expression in adulthood (Church et al., 2021). Considering the consistent findings in both rodent and human studies linking maternal allergies and asthma to offspring behavioral alterations, it stands to reason that specific immunological factors in the maternal environment likely contribute to changes in brain development.

Allergies and asthma are associated with a T-helper type 2 (Th2) immune profile driven by IL-4 signaling (Cho, Stanciu, Holgate, & Johnston, 2005; Wong et al., 2001). Case-control studies have noted elevations in IL-4 in pregnant women during mid-gestation with increased risk of having a child later diagnosed with ASD (Carter, Casey, O'Keeffe, Gibson, & Murray, 2021; Goines et al., 2011). Similarly, asthma-associated cytokines in maternal serum, including IL-4, are correlated with increased risk of ADHD diagnosis in young children (Gustafsson et al., 2020; Thurmann et al., 2019). Rodent studies demonstrate a causal link between individual cytokine concentrations, such as IL-6 and IL-17A, in maternal serum and offspring brain and behavioral development (Choi et al., 2016; Smith, Li, Garbett, Mirnics, & Patterson, 2007). IL-4 signaling is increasingly being recognized as a prominent signaling molecule for higher-order brain function including memory and learning, and IL-4 signaling is proposed to be an important regulator of neuropathology (Gadani, Cronk, Norris, & Kipnis, 2012). For example, IL-4 has neurotrophic properties through its regulation of nerve growth factor synthesis in astrocytes (Awatsuji et al., 1993; Brodie, Goldreich, Haiman, & Kazimirsky, 1998). Moreover, altered IL-4 signaling is observed in several neurological diseases including Alzheimer’s, multiple sclerosis, and glioblastoma (Arababadi, Mosavi, Ravari, Teimori, & Hassanshahi, 2012; Kiyota et al., 2010; Puri, Leland, Kreitman, & Pastan, 1994).

Murine knockout models have been developed to study the role of IL-4 in immune and central nervous system development and behavioral sequelae. Genetic deletion of the IL-4 cytokine and the IL-4 receptor impact several behavioral domains including social interactions, anxiety, mechanical sensitivity, and cognition (Hanuscheck et al., 2022; Moon et al., 2015; Uceyler, Topuzoglu, Schiesser, Hahnenkamp, & Sommer, 2011), suggesting that disruptions to IL-4 signaling during development may impact brain function during later life. Given the influence of immune signaling on brain development (Deverman & Patterson, 2009), and the central role IL-4 plays in the allergic asthma response, it is important to identify whether maternal IL-4 signaling is a necessary cytokine responsible for offspring behavioral deficits in the MAA phenotype.

Considering the central role of IL-4 in MAA, its implications in several developmental disorders, and the previously observed behavioral impacts of MAA on offspring behavior in mice, it is hypothesized that inhibiting IL-4 signaling during pregnancy will mitigate the behavioral impacts of MAA. To test this, we utilized the IL-4 knockout (KO) mouse to determine whether MAA in the absence of IL-4 signaling would still impart behavioral deficits in offspring.

2.0. METHODS

2.1. Animals

Male and female C57Bl/6-Il4^tm1Nnt/J mice (IL-4 KO; stock #002518) and wild type mice C57Bl/6J (C57; stock #000664) were purchased from Jackson Laboratory (Bar Harbor, MA, USA), bred at Mount Holyoke College, South Hadley, MA, and maintained at ambient room temperature on a 12 h light/dark cycle (lights on at 0800 h). Mice were group-housed in individually ventilated cages with same-sex littermates until breeding at 8-weeks of age. Cages were maintained in a temperature-controlled (23° C) vivarium with food and water provided ad libitum. All procedures were performed with approval by the Mount Holyoke College Institutional Animal Care and Use Committee and in accordance with the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.2. Maternal allergic asthma inductions

Allergic asthma inductions were carried out using procedures previously described (Schwartzer et al., 2015; Schwartzer et al., 2017). Sexually-naïve C57 and IL-4 KO female mice were randomly assigned to treatment groups and sensitized with either 50μg ovalbumin (OVA, Sigma, St Louis, MO, USA) and 1mg (Al)OH₃ (Invitrogen, San Diego, CA, USA) dissolved in 200μl of 1X phosphate buffered saline (PBS; Product Number 46-013-CM, Corning Mediatech, Inc, Manassa, VA, USA) or only 1mg (Al)OH₃ dissolved in 200μl of 1X PBS and injected intraperitoneally at 6 and 7 weeks of age (Figure 1A). Beginning at 8 weeks of age, females were mated overnight, presence of seminal plug was checked daily, noted as gestational day (GD)0.5, and single-housed. In alignment with their sensitization treatment group, pregnant mice received either an aerosolized solution of 1% (wt/vl) OVA in PBS or PBS alone for three 45-minute induction sessions occurring at GD 9.5, 12.5, and 17.5, to correspond with early, middle, and late gestation as previously described (Schwartzer et al., 2015). Aerosol inductions were administered via whole-body exposure by placing dams in enclosed mass dosing chambers fitted with Aerogen Pro nebulizer heads (2.4 – 4μm particle) connected to a mass dosing controller (Data Science International, Harvard Bioscience, Inc, Holliston, MA, USA). The treatment and genotype conditions resulted in four maternal exposure groups: C57 PBS (n=12), C57 OVA (n=14), IL-4 KO PBS (n=8), and IL-4 KO OVA (n=7). Additional experimental details in accordance with reporting guidelines proposed by Kentner et al. (2019) can be found in the supplemental methodologies.

Figure 1.

(A) Schematic of the experimental timeline for the maternal allergic asthma exposure paradigm. Four hours after the final aerosol indication, serum was collected from C57 and IL-4KO dams and assessed for (B) IgE and IgG2 antibodies using ELISA, and (C) cytokine concentrations using multiplex bead-based immunoassay. *p <0.05 Bars represent marginal means ±SE. ND: none detected. C57 PBS (n = 9-13), C57 OVA (n=7-14), IL-4 KO PBS (n=6-8), IL-4 KO OVA (n=5-7).

2.3. Anti-OVA IgE and IgG2 ELISA

To assess the allergic immune response to OVA induction with and without the presence of intact IL-4 signaling, anti-OVA IgE and IgG2 antibodies were evaluated in maternal sera collected under isoflurane anesthesia four hours after a final induction using a sandwich ELISA (IgE, Item No. 500840, Cayman Chemicals, Ann Arbor, MI, USA; IgG2, Catalogue No. 3016, Chondrex Inc., Woodinville, WA, USA) with a sensitivity of 3.12 ng/mL and 0.8ng/ml, respectively. All samples were processed in duplicate and unknown quantities were extrapolated from a standard curve using a 4-parameter logistic regression.

2.4. Multiplex bead-based immunoassay

Maternal cytokine concentrations were measured using a 9-plex Procarta Mouse Cytokine kit (EPX170-26087–901, Invitrogen, Waltham, MA, USA) to quantify the following cytokines: IFNγ, IL-4, IL-5, IL-1β, IL-6, IL-13, TNFα, IL-10, and IL-17A. Bead sets were analyzed on a MAGPIX system (Luminex, Austin, TX, USA) using xPONENT 4.1 software according to manufacturer′s instructions. Unknown sample cytokine concentrations were estimated using a 5-parameter logistic regression curve derived from the known reference cytokine concentrations supplied by the manufacturer. Supernatant aliquots did not undergo multiple freeze thaw cycles. The sensitivity of this assay allowed for the detection of cytokine concentration with the following sensitivity: IFNγ: 0.09 pg/mL; IL-13: 0.16 pg/mL; IL-1β: 0.14 pg/mL; IL-4: 0.03 pg/mL; IL-5: 0.32 pg/mL; IL-6: 0.21 pg/mL; TNFα: 0.39 pg/mL; IL-10: 0.69 pg/mL; IL-17A: 0.08 pg/mL.

2.5. Offspring behavioral assessments

A total of 41 dams were used to generate 257 offspring (Table 1) for behavioral testing. Behavioral assessments began at P21 immediately after weaning with the juvenile reciprocal social interaction task. Starting at 8 weeks of age mice were evaluated for approach/avoidance behavior in the elevated plus maze task, general locomotor activity in the open field exploration task, object memory in the novel object recognition task, cognitive appraisal and learned helplessness in the forced swim task, and repetitive motor behaviors in grooming and marble burying tasks. All behavioral assessments were performed between 0800 h and 1200 h by individuals blinded to maternal exposure conditions. Every offspring from each litter completed all behavioral tasks in the same order beginning with the juvenile reciprocal social interaction task and all mice were exposed to no more than one task per week. Individual mice were omitted from a single behavioral task when data were missing due to technical error. A total of 7 mice were lost to attrition due to husbandry and/or human error. A summary of all missing data for each task can be found in Table S1.

Table 1.

Litter Composition.

Genotype	Treatment	Litter, n	Offspring, n		Proportion Male mean (sd)		Litter Size, n mean (sd)		Gestation, days mean (sd)
			Female	Male
C57	PBS	12	45	30	0.52	(0.19)	7.2	(1.56)	19.4	(0.50)
C57	OVA	14	41	45	0.40	(0.28)	5.4	(2.53)	19.1	(0.62)
IL-4KO	PBS	8	20	24	0.53	(0.26)	6.5	(1.51)	19.5	(0.53)
IL-4KO	OVA	7	24	28	0.54	(0.16)	6.3	(1.80)	19.3	(0.49)

2.5.1. Juvenile reciprocal social interaction task

Following weaning at P21, offspring were evaluated for changes in social behavior using the reciprocal social interaction task. Mice were placed individually into clean plastic cages (25×14×12 cm) and allowed to habituate for 20 minutes while being video recorded to monitor locomotor activity. Experimental mice were then quickly returned to their home cage and marked with blue (experimental mice) or pink (sex-, genotype-, and weight-matched stimulus mice) hair chalk (OPAWZ, Ontario, Canada). Experimental and stimulus mice were returned to the arena at opposite ends and allowed to interact for 20 minutes. Mice were video recorded during this time and later scored for the total distance traveled, amount of social sniffing, and body contact behaviors using EthoVision XT 15 three-point body and social interaction modules. Each stimulus mouse was used 2–4 times throughout the study and never more than once per day.

2.5.2. Elevated plus maze

At eight weeks-of-age, offspring were tested for anxiety-associated behaviors in the elevated plus maze. The maze consists of two open (30×5×0.25 cm) and two closed (30×5×6 cm) arms extending from a central platform (5x5 cm) to form a plus shape. The apparatus was elevated 1 m from the floor. Each mouse was placed onto the central platform and allowed 5 minutes of free exploration in the apparatus. Mouse behavior was video recorded under bright lighting and later scored using EthoVision XT 15 for the time spent in each arm. Reductions in the percent of open arm exploration (time in the open arm divided by the total time in both the open and closed arms) were interpreted as increased anxiety.

2.5.3. Open field task

Mice were placed in an open arena (60×42.5×30 cm) and video recorded during 20 minutes of free exploration. Videos were analyzed using EthoVision XT 15 for total distance traveled and the total time spent in the center of the arena.

2.5.4. Novel object recognition

Mice were returned to the open field arena and assessed for memory performance in the novel object recognition (NOR) task using the short habituation protocol (Leger et al., 2013). During the initial familiarization (training) phase, mice were allowed 10 minutes to freely explore two identical objects. Objects used were similar to those described Vogel-Ciernia and Wood (Vogel-Ciernia & Wood, 2014) consisting of square glass votive holders (5 cm x 5 cm x 5 cm) and round tin spice jars (6 cm diameter x 5 cm height) filled with concrete for added weight to limit sliding in the arena. Twenty-four hours later, one of the familiar objects was replaced with a novel object and experimental mice were returned to the arena and video recorded for a 10-minute testing session. Object investigation, as defined by the time spent sniffing either the familiar or novel object, was video recorded and measured using EthoVision XT 15. Novel object recognition, as described by (Lueptow, 2017), was calculated by measuring the time spent sniffing the novel object divided by the total time sniffing either object during the 10-minute trial. Only mice who actively sniffed objects for a minimum of 20 seconds in the familiarization phase were included in the novel object recognition test phase. Novel object sniff times greater than 50% indicated object recognition. All objects and arenas were cleaned with 70% ethanol between each testing session to remove any olfactory cues.

2.5.5. Forced swim test

Mice were placed in a transparent glass cylinder (13 cm diameter × 24 cm high) filled with warm water (22–25° C) to a height of 16 cm. Animals were then recorded for 6 minutes and measured for time spent actively swimming or immobile (i.e., the period of time not spent actively exploring, swimming, or trying to escape) (Can et al., 2012; Porsolt, Le Pichon, & Jalfre, 1977). Measurements were determined using EthoVision XT 15. Following the 6-minute task, mice were removed from the cylinder, toweled dry, and placed in a warmed, dry cage for 20 minutes before being returned to their home cage.

2.5.6. Marble burying

Mice were habituated individually for 10 minutes to opaque plastic mouse cages (25×14×12 cm) filled with a 4-cm thick layer of clean corncob bedding. Following habituation, animals were returned to their home cage and 15 glass marbles were laid out in five rows of three marbles placed equidistant apart. Mice were then returned to their testing cages and allowed to explore for 10 minutes. At the end of the 10-minute period, animals were gently removed from the testing cages and the number of marbles buried was recorded by an observer blinded to treatment condition. Only marbles covered by 75% or more bedding were counted as buried.

2.5.7. Grooming

Mice were placed inside an empty, clear plastic cage and left undisturbed to habituate for 10 minutes in a dimly lit room as previously described (Deacon, 2006). Following the habituation period, mice were video recorded for an additional 10 minutes and later hand-scored for self-grooming behavior by two individuals blinded to the treatment conditions. Grooming was defined as time spent licking paws, washing the nose and face, or scratching fur with any foot. Inter-rater reliability was confirmed using an interclass-correlation coefficient to be greater than 95%.

2.6. Statistical analysis

Data were analyzed using RStudio version 2023.09.1+494 (2023). Anti-OVA IgE and IgG2 antibodies and maternal serum cytokines were analyzed using Kruskal-Wallis rank-sum test followed by pairwise comparisons using Wilcoxon rank sum test with continuity correction from the “stats” package. Litter size, proportion of male mice per litter, and gestational length were assessed using two-way factorial ANOVA (treatment by genotype). To control for pseudoreplication and litter-to-litter variations, offspring measures were evaluated using multilevel modeling as previously described (Church et al., 2021). All behavioral measures were assessed with the “nlme” package using separate linear mixed-effects models for male and female offspring to prevent overfitting and improve statistical power. Each model was fitted using a forward-stepwise regression approach, maximum likelihood estimates, and Type III sums of squares. First, a random-effects only model was constructed with litter set as the random effect. Then, a model containing just the fixed effect for genotype (C57 vs IL-4 KO) was added followed by a model containing both genotype and treatment (PBS vs OVA). A final model was constructed that included both the main effects and the interaction of treatment and genotype. Model fit was assessed using the likelihood ratios test and the best model was selected based on the Akaike Information Criterion (AIC). Model estimates for fixed effects (including 95% confidence intervals and p-values), variance estimates for random effects, and model fit parameters can be found in supplementary materials. For models with significant interaction effects, groups were further analyzed using pairwise comparisons of estimated marginal means with Tukey corrections. For each dependent variable, data were explored for influential outliers using Cook’s distance with a D value greater than 4/n designated as an observation of interest.

3.0. RESULTS

3.1. Maternal serum analysis

Pregnant C57 and IL-4 KO dams sensitized with OVA-Alum or PBS-Alum were challenged with aerosolized OVA or PBS, respectively at GDs 9.5, 12.5, and 17.5. Four hours after a final post-weaning induction, blood was collected and serum concentrations of anti-OVA IgE and IgG2 antibodies were measured using ELISA (Figure 1B). Anti-OVA IgE antibodies were detected in high concentrations in C57 dams exposed to aerosolized OVA compared with genotype-matched PBS dams (p = 0.037) as well as IL-4 KO dams exposed to PBS (p = 0.013) and OVA (p = 0.013). IL-4 KO mice exposed to aerosolized OVA also showed a modest increase in anti-OVA IgE antibodies compared with PBS-exposed IL-4 KO dams (p = 0.013). Given the role of IL-4 in IgG class switching (Faquim-Mauro & Macedo, 2000; Ishizaka et al., 1990) we also examined the impact of aerosolized OVA exposure on IgG2 antibodies. IL-4 KO mice exposed to aerosolized OVA showed significant elevations in IgG2 concentration compared with both IL-4 KO mice exposed to PBS (p = 0.009) and C57 mice exposed to either PBS (p = 0.005) or OVA (p = 0.008). IgG2 levels were also elevated in C57 dams exposed to OVA compared with genotype-matched PBS-exposed dams (p = 0.001), but at a lower concentration compared with IL-4 KO dams treated with OVA (p = 0.008).

Maternal serum cytokine concentrations were measured using multiplex bead-based immunoassay (Figure 1C). C57 dams exposed to aerosolized OVA showed increases in the allergic-asthma associated cytokine IL-4 compared with C57 dams exposed to PBS. Conversely, IL-4 concentrations were below the limit of detection for both PBS and OVA-exposed IL-4 KO mice. These increases in IL-4 concentrations in OVA-exposed C57 mice were mirrored by significant increases in IL-5 (p = 0.023) and marginal increases in IL-6 (p = 0.058) compared with C57 dams exposed to PBS. The concentration of IL-5 or IL-6 in IL-4 KO mice of either treatment group was no different from C57 PBS dams (p > 0.05 for all comparisons) (Figure 1C). No differences were detected in any other cytokines measured between genotype or treatment groups.

3.2. Litter composition and gestational length

The total number of pups per litter, proportion of male offspring, and gestational length for each dam was assessed between genotype and treatment using two-way factorial ANOVA (Table 1). There was a significant effect of treatment on litter size, F(1, 37) =5.35, p = 0.026, with OVA-exposed dams having an average of 5.7 mice per litter compared to 6.9 mice per litter in PBS-exposed dams. Post hoc analysis confirmed that these differences in litter size between MAA and control dams were present in C57 litters (p = 0.026) but not IL-4 KO litters (p = 0836). No differences were observed in litter size between genotypes, F(1,37) = 0.54, p = 0.467. There were no additional effects of treatment on the proportion of male offspring, F(1, 37) = 1.64, p = 0.209 and no differences between genotype, F(1, 37) = 0.01, p = 0.907. The average gestational length was 19.275 days and no differences were observed between treatment groups, F(1, 37) = 1.49, p = 0.230 or genotype, F(1, 37) = 0.72, p = 0.402.

3.3. Social interactions

Following weaning, juvenile offspring were allowed to acclimate for 20 minutes in an open arena followed immediately by a 20-minute social interaction with a novel C57 stimulus mouse (Figure 2A). In female offspring, no differences were observed between genotype or treatment for any social interaction measures. A random effects-only model was best suited for all behavioral outcomes (Table S2-S6). In males, a model containing both genotype and treatment was selected for total social sniffing time, χ²(1) = 5.04, p = 0.025 , as well as nose-to-body measurements, χ²(1) = 5.33, p = 0.021 (Table S2, S4). For total social time, there was a significant effect of treatment on C57 offspring compared with genotype-matched PBS controls (p = 0.027). On average, C57 offspring of PBS-treated dams spent 208 seconds sniffing a novel mouse (95% CI: 166.12 – 249.92). Conversely, offspring of MAA dams spent an average of 59.26 seconds less (95% CI: 7.96 – 110.57) engaging in social sniffing behaviors compared with PBS controls (Figure 2B). These lower levels of social sniffing were not observed in male IL-4 KO offspring from either PBS- or OVA-treated dams compared to C57 offspring of PBS-exposed dams (p = 0.277).

Figure 2.

Juvenile, 3-week-old, offspring were assessed for social behaviors in the reciprocal social interaction task. (A) Offspring were allowed to habituate for 20 minutes in an empty arena followed immediately by the introduction of a novel weight-, genotype-, and sex-matched C57 stimulus mouse. (B) Social sniffing, (C) close body contact, and (D) distance traveled were measured automatically using EthoVision XT 15 software over 20 minutes. *p < 0.05 as determined by linear mixed-effects modeling with treatment and genotype as fixed effects and litters as random effects. Plots represent individual mice; bars represent marginal means ± SE.

In addition to social sniffing, mice were measured for broader social proximity including huddling behavior defined by total body contact. A random effects-only model was selected for female mice, with no differences observed in total body contact time between genotype or treatment groups. In males, however, a model containing genotype, treatment, and their interaction as fixed effects was selected, χ²(2) = 6.27, p = 0.044. There was a significant effect of treatment (p = 0.016) on total body contact time among male offspring (Table S6). Compared with C57 PBS mice, C57 offspring born from OVA-exposed dams had a 111 second reduction (95% CI: 23.62 – 198.28) in total body contact time during the 20-minute social interaction task (p = 0.016). Post hoc analysis further confirmed these lower levels of huddling behavior in C57 OVA offspring compared with C57 PBS offspring (p = 0.07) and IL-4 KO offspring of both PBS (p = 0.046) and OVA dams (p = 0.040) (Figure 2C). The mixed-effects model did not identify a significant main effect for genotype (p = 0.675). IL-4 KO mice born from PBS-treated dams engaged in similar amounts of body contact time compared to C57 PBS offspring (p = 0.974) and no effect of maternal OVA exposure was observed in IL-4 KO offspring compared to C57 PBS mice (p = 0.960) and genotype-matched IL-4 KO PBS offspring (p = 0.999).

While the impact of MAA on offspring social measures were only observed in C57 offspring, there were other genotype-specific differences detected in the overall motor activity between IL-4 KO and C57 offspring. For both male and female mice, a mixed-effects model containing genotype as a fixed effect was selected for the total distance traveled during the 20-minute social interaction task, male, χ²(1) = 11.93, p <0.001; female, χ²(1) = 4.74, p = 0.029 (Table S7). Specifically, IL-4 KO mice of both sexes had a significantly lower level of total distance traveled during the social interaction task compared with C57 offspring (female, p = 0.029; male, p = 0.001) (Figure 2D).

3.4. Approach/avoidance behavior

In the elevated plus maze, exploration of the open arms was standardized by dividing the time spent in the open arms by the total time spent in both open and closed arms. The time in the center of the arena was excluded from the analysis (Figure 3A). A genotype-only model was selected for both female and male mice for the percent time in the open arms of the arena, female, χ²(1) = 8.27, p = 0.004; male, χ²(1) = 6.31, p = 0.012 (Table S8). Compared with sex-matched C57 offspring, IL-4 KO mice, regardless of treatment, spent a lower percentage of time on the open arms of the elevated plus maze. For IL-4 KO mice, males showed an average of 9.15% reduction in open arm exploration (95% CI: 1.50 - 16.80, p = 0.021) and females displayed an average of 11.52% reduction (95% CI: 4.27 - 18.78, p = 0.003), compared with C57 offspring (Figure 3B).

Figure 3.

At 8 weeks of age, offspring were assessed for approach/avoidance behaviors and locomotor activity in the elevated plus maze and open field task, respectively. (A) Mice were allowed to explore the elevated plus maze for 5 minutes and measured for time spent in the open and closed arms of the maze using EthoVision XT 15 software. (B) Percent time in the open arms was calculated as the time spent in the open arms divided by the total time spent in open and closed arms. (C) Latency to first enter the open arm was assessed as the first instance the mouse crossed into the open arms of the arena. (D) Locomotor activity during the elevated plus maze was assessed as total distance traveled. (E) In the open field task mice were allowed 20 minutes to explore a novel arena and measured for (F) the total time in the center of the arena, (G) the latency to first enter the center, and (H) total distance traveled. ^#p < 0.10 *p < 0.05 as determined by linear mixed-effects modeling with treatment and genotype as fixed effects and litters as random effects. Plots represent individual mice; bars represent marginal means ± SE.

In addition to differences in percent open arm exploration, there was an effect of genotype on the latency to first enter the open arm of the elevated plus maze. In male offspring, a genotype-only model identified a significant difference between IL-4KO and C57 mice in open arm latency, χ²(1) = 4.90, p = 0.027. On average, IL-4KO mice took 4.80 seconds (95% CI: 0.51 – 9.10, p = 0.031) longer to first enter the open arm of the maze compared to sex-matched C57 offspring (Figure 3C). While female offspring showed similar differences in latency between genotype the mixed effects model did not reach statistical significance χ²(1) = 3.18, p = 0.074 (Table S9).

To determine whether these differences in elevated plus maze exploration were impacted by locomotor differences, the total distance traveled on the maze was measured for each mouse. A mixed-effects model containing genotype, treatment, and their interaction was selected for female offspring, χ²(1) = 13.02 p <0.001 (Table S10). The model revealed a significant treatment by genotype interaction for total distance traveled (p < 0.001). Specifically, female C57 offspring of OVA-exposed dams showed an increase in total distance traveled compared with sex-matched offspring of PBS-exposed C57 dams (p = 0.026). However, this increase in motor activity was not present in IL-4 KO offspring of PBS-treated (p = 0.759) nor OVA-treated dams (p = 0.107) compared to C57 PBS offspring (Figure 3D). No effect of maternal OVA exposure was observed in IL-4 KO offspring (p = 0.491). In males, there was no observed difference in distance traveled between any groups as indicated by a random-effects only model (Table S10).

3.5. General locomotor activity

In the open field task (Figure 3E), no differences were observed across genotype or treatment for the total time spent in the center (Figure 3F) and the latency to first enter the center (Figure 3G) of the open field arena for either sex (Table S11-12). In female mice, a genotype-only model was selected for total distance traveled, χ²(1) = 10.41 p = 0.001. IL-4KO female mice traveled an average of 896.68cm (95% CI: 376.45 – 1416.90, p = 0.001) less compared with sex-matched C57 offspring (Figure 3H). These genotype differences in locomotor activity were not observed in male mice (Table S13).

3.6. Cognitive appraisal and learned helplessness

Offspring were assessed for differences in immobility time during the 6-minute forced swim task (Figure 4A). For both male and female mice, a mixed-effects model was selected containing both treatment, genotype, and their interaction, male, χ²(1) = 7.42, p = 0.006; female, χ²(1) = 7.10, p = 0.008 (Table S14). In male offspring, there was a significant effect of genotype (p < 0.001) with IL-4 KO mice spending an average of 34 fewer seconds (95% CI: 17.00 – 52.03) in the immobile position compared with C57 mice. In addition, there was a significant genotype by treatment interaction (p = 0.010), with IL-4 KO offspring born from OVA-treated dams showing higher immobility time compared with PBS-treated IL-4 KO offspring (Figure 4B). Post hoc analysis confirmed significantly longer immobility times in IL-4 KO offspring of OVA-treated dams compared with genotype-matched PBS offspring (p < 0.001). This impact of OVA exposure during pregnancy on IL-4 KO offspring was not present in C57 offspring. While the mixed-effects model identified a marginal effect of treatment on immobility time (p = 0.065), post hoc analysis did not reveal an increase in immobility time in C57 OVA offspring compared to C57 PBS control mice (p = 0.243). Similar to male offspring, females displayed a genotype by treatment interaction in immobility measures. There was a significant effect of genotype (p = 0.002) with IL-4 KO female offspring born from PBS-treated dams spending approximately 41 fewer seconds (95% CI: 17.23 – 64.61) immobile compared with C57 female offspring, and these reductions in immobility time were not present in female IL-4 KO mice born from OVA-treated dams as indicated by a treatment by genotype interaction (p = 0.010). Post hoc analysis confirmed that female IL-4 KO mice born from OVA-exposed dams showed immobility scores similar to sex-matched C57 offspring of both PBS-exposed dams (p = 0.842) and OVA-exposed dams (p = 0.967). Finally, the effect of maternal OVA exposure on female C57 offspring compared to genotype-matched PBS controls was not significant (p = 0.967).

Figure 4.

(A) Adult offspring underwent a 6-minute forced swim task and were assessed for (B) total time spent immobile. (C) Memory performance was measured using the novel object recognition (NOR) task. Following habituation, mice were given 10 minutes to freely explore 2 identical objects. Twenty-four hours later, mice were reintroduced to the arena with one object replaced by a novel object and allowed 10 minutes of exploration. (D) NOR score was calculated as the time spent sniffing the familiar object divided by the total time sniffing the familiar and novel objects. (E) Object memory was evaluated as spending significantly more time with the novel object compared with familiar object. *p < 0.05 as determined by linear mixed-effects modeling with treatment and genotype as fixed effects and litters as random effects. For object sniff measures, linear mixed-effects modeling included object, treatment, and genotype as fixed effects and litters and animal as random effects. Plots represent individual mice; bars represent marginal means ± SE.

3.7. Object memory

Memory performance was tested in the NOR task. Twenty-four hours after an initial exposure to two identical objects, one object was replaced with a novel object and mice were given 10 minutes to explore both the familiar and novel objects (Figure 4C). Object exploration times were standardized across animals by calculating a NOR score equal to the time spent sniffing the novel object divided by total object sniff time. For both male and female mice, a mixed-effects model identified a significant effect of genotype on object recognition scores male, χ²(1) = 8.54, p = 0.003; female, χ²(1) = 13.11, p < 0.001(Table S15). In males, IL-4 KO mice scored an estimated 4.86 (95% CI: 0.74 – 8.98) higher on the NOR task compared with C57 offspring (p = 0.023), with similar increases also observed in female IL-4 KO mice (95% CI: 3.55 – 10.82, p < 0.001) (Figure 4D). These differences in NOR performance between genotype were driven by deficits in object memory formation in C57 offspring born to MAA-exposed dams (Figure 4E). Specifically, a mixed-effects analysis comparing sniff times for novel object versus familiar object revealed a significant treatment by genotype interaction, χ²(1) = 13.24, p = 0.01 (Table S16). While C57 offspring born to PBS-exposed dams showed a significant preference for the novel object (p = 0.006), offspring of OVA-exposed C57 dams had similar sniff times between novel and familiar object (p = 0.539). Conversely, IL-4 KO offspring from both PBS- and OVA-exposed dams spent significantly more time sniffing the novel object compared with familiar object (IL-4 KO PBS, p < 0.001; IL-4 KO OVA, p < 0.001).

3.8. Repetitive motor behaviors

Assessment of grooming behavior during a 10-minute cage observation (Figure 5A), identified a significant effect of genotype in both male, χ²(1) = 11.66, p <0.001, and female, χ²(1) = 8.18, p = 0.004 mice (Figure 5B). Male IL-4 KO mice spent an estimated 23.21 fewer seconds (95% CI: 9.83 – 36.59) grooming during the 10-minute observation compared with C57 offspring. Similarly, IL-4 KO female mice spent an average of 21.84 fewer seconds (95% CI: 7.33 – 21.84) compared with sex-matched C57 mice. Mixed-effects models containing treatment or genotype by treatment interactions were not significant, indicating no effect of maternal OVA exposure on grooming behavior in either genotype (Table S17).

Figure 5.

(A) Grooming behaviors were assessed during a 10-minute recorded observation. (B) Total time spent grooming was assessed by two researchers blinded to treatment condition and analyzed using linear mixed-effects modeling with treatment and genotype as fixed effects and litters and animal as random effects. (C) In the marble burying task, mice were allowed 10 minutes to habituate to a clean cage filled with corn cob bedding. After habituation, 15 marbles were lined up in 5 rows of 3 marbles and mice were given 10 minutes of free exploration. (D) The total number of marbles buried were counted by two observers blinded to treatment condition and analyzed using a negative binomial model to account for overdispersion. *p <0.05. Plots represent individual mice; bars represent marginal means ± SE, violin plots represent frequency densities.

In the marble burying task a similar effect of genotype was observed in the absence of any effect of maternal OVA exposure, male, χ²(1) = 3.21, p = 0.07; female, χ²(1) = 11.93, p = 0.032 (Figure 5C,D). Using a negative binomial model to account for mice that failed to bury marbles, there was a significant effect of genotype for females (p = 0.025) and males (p = 0.049). Female IL-4 KO mice buried an average of 2 more marbles compared with sex-matched C57 mice (95% CI: 1.12 - 5.23). While the final statistical model for males did not reach significance (p = 0.073), the trend suggests a similar 1.77 marble increase for IL-4 KO mice compared with male C57 offspring (95% CI: 1.00 – 3.11). Models containing treatment and a treatment by genotype interaction were not significant suggesting no effect of OVA exposure on offspring of either genotype (Table S18).

4.0. DISCUSSION

The maternal immune environment plays an influential role in shaping fetal brain development and risk for neurodevelopmental disorders such as ASD and ADHD. Allergic asthma during pregnancy and maternal IL-4 serum levels are associated with increased risk of ASD and ADHD, and rodent studies have demonstrated a direct impact of IL-4 signaling on myelination, pruning, and brain networking (Guedes et al., 2023; Hanuscheck et al., 2022; Zanno et al., 2019). To investigate the effects of IL-4 signaling on offspring development, we tested whether the behavioral effects of MAA are present in an IL-4 deficient mouse. While maternal OVA exposure in C57 dams reduced social interaction in male offspring and impaired NOR task performance in both sexes, IL-4 deficient offspring of MAA dams exhibited lower motor activity in the open field, reduced grooming, and lower rates of open arm exploration in the elevated plus maze. The phenotypic differences in the IL-4 KO mouse, coupled with the IL-4-dependent changes observed in MAA offspring, highlight an important developmental role for IL-4 signaling in species-typical behavioral processes.

Analysis of maternal serum in our OVA-exposed dams confirmed the presence of Th2-mediated allergic asthma inflammation in C57 mice, indicated by elevated levels of IL-4, IL-5, and IL-6. In line with reports demonstrating the importance of IL-4 in allergic asthma inflammation (Hamelmann et al., 2000; Herrick, MacLeod, Glusac, Tigelaar, & Bottomly, 2000; Kropf & Mueller, 2003), IL-4, IL-5, and IL-6 were not elevated in the serum of IL-4 KO dams. Similarly, OVA exposure produced a high concentration of anti-OVA IgE antibodies in C57 dams and much smaller increases in OVA-exposed IL-4 KO dams, underscoring the essential role of IL-4 in regulating the Th2/IgE allergy response to OVA (Brusselle et al., 1994). In the absence of IL-4 the antibodies made by plasma cells are predominantly IgG2a and IgG2b (Faquim-Mauro & Macedo, 2000). In line with this work, we observed blunted levels of IgE and heightened concentrations of IgG2 antibodies in response to OVA allergen exposure in the IL-4 KO mice. These findings indicate that IL-4 KO mice mount an immunological response to the MAA paradigm through an IL-4-independent mechanism. This alternate mechanism may pose unique developmental effects on offspring typically dampened in the presence of elevated IL-4.

During development, IL-4 modulates pruning in the cerebellum which can have downstream effects on motor activity and coordination (Guedes et al., 2023). In the IL-4 KO mouse, we observed altered motor behaviors in open field, grooming, and marble burying tasks suggestive of a hypomotor state. Male and female mice lacking the IL-4 cytokine traveled less in the open field task, buried more marbles, and engaged in less time grooming compared to wild type mice regardless of maternal OVA exposure. Elevated levels of IL-4 during periods of brain development in mice impart a hyper-locomotor phenotype in later life, and these behaviors can be mirrored through OVA allergen-induced release of IL-4 in the early postnatal period (Guedes et al., 2023). Therefore, the lower levels of locomotor activity observed in our IL-4 KO mice may reflect an opposing effect of IL-4 deficiency during these developmental processes. The lower grooming rates in IL-4 KO mice may also support this link between IL-4 signaling and motor activity development, or it may reflect greater sensory-motor sensitivity through mechanical hypersensitivity (Uceyler et al., 2011).

In addition to these motor differences, mice lacking IL-4 showed altered behavioral outcomes in tasks associated with anxiety-like behaviors. Specifically, IL-4 KO mice spent less time in the open arms and longer latency to first enter the open arm of the elevated plus maze compared with C57 mice. Conversely, no differences were observed between genotypes in center exploration time of the open field arena. Open field exploration times are typically lower in mice who show low levels of open arm exploration in the elevated plus maze (Carola, D'Olimpio, Brunamonti, Mangia, & Renzi, 2002), and these two behavioral measures often correlate in response to chronic stress and anxiety (La-Vu, Tobias, Schuette, & Adhikari, 2020), though not consistently (Figueiredo Cerqueira et al., 2023). Importantly, these differences in open arm exploration were not observed in response to maternal OVA exposure. That is, IL-4 KO and C57 offspring born from MAA dams did not exhibit differences in open arm exploration in the elevated plus maze compared to genotype-matched control offspring, a finding that parallels earlier maternal allergic inflammation work in both mice and rats (Breach et al., 2021; Schwartzer et al., 2017).

The forced swim task is a measure of stress reactivity that provides context for cognitive evaluation and coping behaviors (de Kloet & Molendijk, 2016; Molendijk & de Kloet, 2015, 2019). IL-4 KO mice born from PBS-exposed dams spent less time in the immobile position compared to C57 mice. This is in contrast to Moon et al (2015) who reported no differences in the forced swim task in the IL-4 KO mice and Wachholtz et al. (2017) who reported increased immobility time (Moon et al., 2015; Wachholz et al., 2017). Importantly, Wachholtz’s group used an IL-4 KO mouse on a BALB/c background limiting direct phenotypic comparisons to our IL-4 KO mice on a C57 background. In a model of chronic mild stress, hippocampal IL-4 concentrations negatively correlated with immobility time in the forced swim task and positively correlated in the sucrose preference tests (Zhang et al., 2021). Our observed reduction in immobility time in the IL-4 KO mouse along with the decreased open-arm exploration in the elevated plus maze highlight a role for IL-4 in stress responsiveness, adding to existing work indicating hippocampal IL-4 signaling regulates stress reactivity and spatial learning/memory via neurogenesis modulation (Gadani et al., 2012). It is important to note that these reductions in immobility time were not present in IL-4 KO mice born from OVA-exposed dams, demonstrating an IL-4- and IgE-independent developmental impact of maternal OVA exposure. In fact, we observed a blunted IgE response to OVA in IL-4 KO dams with concomitant elevations in IgG2. While it remains unknown all cytokines that mediate B cell class switching from IgE to IgG2 in IL-4 KO mice, it is plausible that these same signals during pregnancy impact offspring stress reactivity uniquely in the IL-4 KO mouse through IgE-independent mechanisms.

Our findings indicate IL-4 signaling is necessary for maternal OVA exposure to impact juvenile offspring social interaction. Male C57 offspring of MAA dams spent less time engaging in social sniff behaviors and displayed lower rates of huddling behavior compared to offspring of PBS-exposed dams. This finding is consistent with our previous reports of altered social interactions using the same MAA model (Schwartzer et al., 2017) and mirrors observations of reduced juvenile play behavior in a rat model of maternal allergic inflammation (Breach et al., 2021). Interestingly, IL-4 KO mice did not show any phenotypic differences in social interactions compared to C57 mice born from control dams, which contrasts with Moon et al.’s findings of increased social sniff time in adult IL-4 KO mice (Moon et al. 2015). Important methodological differences, including testing adult experimental mice against juvenile conspecifics, limiting social interactions to 5 minutes, and separating dyads by a wire mesh, likely contribute to our contrasting results. In the present study, IL-4 KO mice were assessed during a 20-minute free social exploration task during their juvenile age, a time when social interactions are not directly driven by reproductive, territorial, or parenting processes (Panksepp & Lahvis, 2007; Pellis, Field, & Whishaw, 1999). It is possible that differences in social engagement levels in the IL-4 KO mouse emerge in adulthood when social motivation is driven by post-pubertal processes. These differences notwithstanding, the decreased social engagement observed in the C57 offspring of MAA dams parallel clinical reports linking maternal allergies and asthma with offspring social behavior deficits (Patel et al., 2018) and raise questions about the role of IL-4 in social-emotional brain networks.

IL-4 signaling plays a critical role in cognitive processes related to learning and memory, and studies in mice underscore the importance of meningeal IL-4 signaling in the Morris water maze task (Derecki et al., 2010). Derecki et al. (2010) demonstrated that depletion of IL-4 impairs Morris water maze performance in SCID and B6.129 KO mice driven by meningeal T-cell signaling. Despite our differences in background strain and methodology, we also observed genotypic differences in cognitive performance between IL-4 KO and C57 mice, in particular, NOR score. Moreover, MAA exposure impaired NOR in C57 offspring, but not IL-4 KO mice, suggesting that maternal changes in IL-4 signaling can impact offspring memory performance in adulthood. There is limited research in humans investigating the impact of maternal asthma on offspring cognitive development later in life. One systematic review noted a weak relationship between maternal asthma during pregnancy and offspring neurocognitive development and emphasized the importance of asthma management in mitigating poor offspring outcomes (Whalen et al., 2019). However, the availability of strong prospective reports on allergic asthma severity, symptom management, and offspring cognitive outcomes is limited, so our observation in mice provides initial evidence that object memory performance in response to MAA may be IL-4-dependent.

The observed impacts of MAA and IL-4 deficiency on offspring behaviors are not without limitations. MAA resulted in a significant reduction in litter size in C57 dams without changes in gestational length, but IL-4 KO dams had similar litter sizes regardless of treatment. These differences in litter size highlight a potential consequence of MAA on offspring viability and complicate our ability to elucidate the impact of the prenatal maternal immune environment versus the postnatal rearing period regarding shaping offspring behavior. In C57 mice, a smaller litter size is associated with decreased anxiety and lower stress responsiveness in male offspring (Salari, Samadi, Homberg, & Kosari-Nasab, 2018). However, this was not observed in the C57 mice included here. A limitation of our study design is that the use of homozygous IL-4 KO mice limits our ability to decouple the fetus’ response to OVA exposure from the dam’s, considering that both possess IL-4 deficient alleles. Subsequent studies incorporating heterozygous breeders would allow for genetic variability in the offspring (or dams) to better dissociate the role of maternal IL-4 in shaping MAA-induced offspring behavior from the independent role of fetal IL-4. The inherent complexity in these maternal-fetal exposure experiments also require careful consideration of the analytical approaches used to evaluate offspring outcomes. The use of linear mixed-effects modeling mitigates some of these confounds by using the dam as the experimental unit to control for the litter-to-litter variability (Lazic & Essioux, 2013). This results in a considerably smaller sample size relative to the number of offspring assessed and the potential for a decrease in statistical power. In order to control for type II error, we utilized separate regression models for male and female offspring. This presents its own limitations in data interpretation given that sex-specific effects cannot be inferred statistically. These constraints notwithstanding, the behavioral outcomes highlight important impacts of both MAA and IL-4 on brain development.

4.1. Conclusions

The social and cognitive differences observed in C57 offspring born from MAA dams and the absence of these behavioral observations in the IL-4 KO mice implicate elevated maternal IL-4 signaling in linking allergic asthma during pregnancy to offspring neurodevelopment. Moreover, the altered motor activity observed in IL-4-deficient mice underscores the important role of IL-4 in brain development and subsequent species-typical behavioral outcomes. Future work that targets transient disruptions in IL-4 signaling during pregnancy, as opposed to the sustained knockout afforded in the IL-4-deficient mutant mouse, will help identify specific developmental windows and other immune mechanisms that link maternal allergic inflammation to offspring behavioral changes in later life.

Highlights.

• IL-4 deficient mice have lower IgE-mediated allergic responses and increased IgG2

• Maternal asthma impacts social behavior and cognition in C57 male offspring

• IL-4 knockout mice do not show developmental effect of maternal allergic asthma

• Altered motor activity is observed in IL-4 knockout mice across behavior tasks