Background: Maternal allergies and asthma during pregnancy have been associated with increased risk of ASD and ADHD to the child. Previous rodent studies have demonstrated that inducing a T helper-2 (Th2)-mediated allergic response during pregnancy leads to an offspring behavioral phenotype characterized by decreased social interaction and increased stereotypies. Interleukin-4 is a key signal in the Th2 immune cascade, but its role in fetal brain development and subsequent impacts of maternal allergic asthma (MAA) on offspring behavioral deficits have yet to be determined.

Objective: In this study, we investigated whether the absence of IL-4 signaling would mitigate MAA-induced behavioral changes.

Methods: C57BL/6J and Interleukin-4 knockout (IL-4 KO) mice were sensitized to ovalbumin and exposed to repeated allergic asthma aerosol inductions throughout pregnancy. Offspring were assessed on Juvenile Reciprocal Social Interaction, Elevated Plus Maze, Open Field Exploration, Novel Object Recognition, Forced Swim, Marble-burying, and Grooming tasks.

Results: MAA during pregnancy resulted in decreased social interactions in male C57 offspring and impaired memory performance in both male and female C57 mice. These deficits were not observed in IL-4 KO mice exposed to MAA. However, we observed genotype effects in IL-4 KO mice including altered motor performance and anxiety-associated responses.

Conclusion: MAA-induced social and cognitive behavioral alterations are IL-4 dependent. IL-4KO offspring display genotype-specific differences suggesting IL-4 signaling is important for typical developmental processes.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%.

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.