DOI: 10.5061/dryad.m0cfxpp91
title: Data from: Offspring behavioral outcomes following maternal allergic asthma in the IL-4-deficient mouse

description: 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.
description: <strong>Animals</strong>
             Male and female
 C57Bl/6-Il4<sup>tm1Nnt</sup>/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 <em>ad libitum</em>. 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.
 <strong>Maternal allergic asthma
 inductions</strong>             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)OH<sub>3</sub> (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)OH<sub>3</sub> 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.
 <strong>Anti-OVA IgE </strong><strong>and IgG2
 </strong><strong>ELISA </strong>
             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.
 <strong>Multiplex bead-based
 immunoassay</strong>             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. <strong>Offspring
 behavioral assessments </strong>             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. <strong>Juvenile reciprocal social
 interaction task</strong>             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. <strong>Elevated plus
 maze</strong>             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. <strong>Open
 field task</strong>             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. <strong>Novel object
 recognition</strong>             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. 
 <strong>Forced s</strong><strong>wim
 </strong><strong>test</strong> 
             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.
 <strong>Marble burying </strong>
             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.
 <strong>Grooming</strong>
             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%. <strong>Statistical
 analysis</strong> <strong>           
 </strong>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 <em>D</em> value greater than 4/n
 designated as an observation of interest.
description: # Offspring behavioral outcomes following maternal allergic asthma in the
 IL-4-deficient mouse GENERAL INFORMATION 1. Title of Dataset: Maternal
 serum analysis and offspring behavioral outcomes following maternal
 allergic asthma in C57Bl/6J and IL-4 deficient mince. 2. Author
 Information Corresponding Author Name: Jared Schwartzer Institution: Mount
 Holyoke College Address: 50 College Street, South Hadley, MA USA Email:
 [jjschwar@mtholyoke.edu](mailto:jjschwar@mtholyoke.edu) 3. Data of data
 collection: 2021-2023 4. Funding Source: National Institutes of Health,
 R15MH119500. DATA & FILE OVERVIEW 1. File List: A) Behavior Data.csv
 B) Maternal Serum.csv 1. Relationship between files, if important:
 Maternal Serum ID is Litter ID on Behavior Data.csv 2. Additional related
 data collected that was not included in the current data package: None 3.
 Abbreviations in Datasets JRSI - Juvenile Reciprocal Social Interaction
 EPM - Elevated Plus Maze OF - Open Field NOR - Novel Object Recognition
 FST - Forced Swim Task 4. Definition of Variables **Behavior Data**\
 AnimalID: Animal Identification Number\ Genotype: C57 wild type or IL-4
 Knock out mouse\ Sex: Male or Female\ Litter: Identification Number of the
 dam\ Treatment: Exposure group for phosphate buffered saline control (PBS)
 or Ovalbumin (OVA) aerosol exposure\ DistanceJRSI: Total distance traveled
 (cm) in the juvenile reciprocal social interaction task\ VelocityJRSI:
 Mean velocity cm/s in the juvenile reciprocal social interaction task\
 NosetoNose.JRSI: Total time (seconds) mouse nose was near stimulus mouse
 nose\ NosetoBody.JRSI: Total time (seconds) mouse nose was near central
 body point of stimulus mouse\ NostetoTail.JRSI: Total time (seconds) mouse
 nose was near stimulus mouse's base of tail\ TotalSocial.JRIS: Total
 sum of time (seconds) of NosetoNose, NosetoBody, and NosetoTail\
 BodyContact.JRSI: Total time (seconds) mouse body points were in proximity
 to stimulus mouse\ DistanceEPM: Total distance traveled (cm) in the
 elevated plus maze task\ VelocityEPM: Mean velocity cm/s in the elevated
 plus maze task\ CenterTime.EPM: Total time (seconds) mouse center point
 was in the center of the EPM arena\ OpenTime.EPM: Total time (seconds)
 mouse center point was in the open arms of the EPM arena\ ClosedTime.EPM:
 Total time (seconds) mouse center point was in the closed arms of the EPM
 arena\ LatencyOpen.EPM: Latency to first enter open arm of the EPM arena\
 PercentOpen.EPM: Calculated as OpenTime.EPM/(OpenTime.EPM +
 ClosedTime.EPM)*100\ Distance.OF: Total distance traveled (cm) in the Open
 Field arena\ Velocity.OF: Total velocity cm/s in the Open Field arena\
 Time.Center.OF: Total time (seconds) in the center of the Open Field
 arena\ LatencyCenter.OF: Latency to first enter the center of the Open
 Field arena\ FamiliarTime.NOR: Total time (seconds) the nose point was in
 contact with the familiar object\ NovelTime.NOR: Total time (seconds) the
 nose point was in contact with the novel object\ NOR.Score: Calculated as
 NovelTime.NOR/(NovelTime.NOR+FamiliarTime.NOR)*100\ Left.Object.Time:
 Total time (seconds) sniffing the left object during the training phase of
 the NOR task\ Right.Object.Time: Total time (seconds) sniffing the right
 object during the training phase of the NOR task\ Score1.Grooming: Total
 time (seconds) spent grooming scored by researcher 1\ Score2.Grooming:
 Total time (seconds) spent grooming scored by researcher 2\ Grooming: Mean
 time (seconds) from researcher 1 and research 2 for the time spent
 grooming\ Immobile.FST: Total time (seconds) in low activity immobility
 during the forced swim task\ Marbles: Number of marbles buried **Maternal
 Serum**\ AnimalID: Animal Identification Number of pregnant dam\ Genotype:
 C57 wild type or IL-4 Knock out mouse\ PrimeOnly: Indicates animal
 received aerosol inductions (yes = initial priming only; no = priming and
 aerosol\ IL.10: Interleukin-10 concentration in pg/ml\ IL.1B:
 Interleukin-1 beta concentration in pg/ml\ IL.4: Interleukin-4
 concentration in pg/ml\ IL.5: Interleukin-5 concentration in pg/ml\ IL.6:
 Interleukin-6 concentration in pg/ml\ IL.13: Interleukin-13 concentration
 in pg/ml\ IFNy: Interferon-gamma concentration in pg/ml\ TNFa: Tumor
 Necrosis Factor alpha concentration in pg/ml\ IL.17A: Interleukin-17A
 concentration in pg/ml\ IgG2: Anti-OVA Immunoglobulin-G2b concentration in
 ng/ml\ IgE: Anti-OVA Immunoglobulin-E concentration in ng/ml 5.
 Description of NA Cells with NA are produced when data was not collected
 due to technical error. For Behavior Data NA's indicate the mouse was
 not run through the behavior task or the video capture system was not
 turned on during data collection. 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 of the corresponding manuscript. For Maternal Serum NA indicates
 insufficient bead count (&lt;50 beads read) or insufficient serum
 available to collect data. CODE/SOFTWARE R file represent the code
 structure used to analyze the .CSV scientific data files. Individual
 variables need to be input into statistical models to get outputs that
 reflect those reported in the associate manuscript. The .PDF files
 represent the procedures used to acquire the scientific data.
 SHARING/ACCESS INFORMATION
 [https://doi.org/10.5061/dryad.m0cfxpp91](https://doi.org/10.5061/dryad.m0cfxpp91) Behavior data were generated from Ethovision XT 15, Noldus Information Technology, [https://www.noldus.com/ethovision-xt](https://www.noldus.com/ethovision-xt) Maternal cytokine data were generated from MagPIX Instrument, Luminex Corporation | A DiaSorin Company, and derived using xPonent 4.3 software, [https://int.diasorin.com/en/licensed-technologies/reagents-accessories/software](https://int.diasorin.com/en/licensed-technologies/reagents-accessories/software) UPDATES SINCE LAST VERSION 1. The manuscript associated with these data has moved from 'manuscript in progress' to 'published article' and DOI has been included. 2. Behavior_Data.csv file has been updated in response to peer-review comments. Two additional variables have been added (LatencyOpen.EPM and LatencyCenter.OF). 3. Missing data for Open Field measures were acquired and added to Behavior_Data.csv file.