Altered fear behavior in aeroallergen house dust mite exposed C57Bl/6 mice: a model of Th2-skewed airway inflammation

Abstract

There is a growing interest for studying the impact of chronic inflammation, particularly lung inflammation, on the brain and behavior. This includes asthma, a chronic inflammatory condition, that has been associated with psychiatric conditions such as posttraumatic stress disorder (PTSD). Although asthma is driven by elevated production of Th2 cytokines (IL-4, IL-5 and IL-13), which drive asthma symptomology, recent work demonstrates that concomitant Th1 or Th17 cytokine production can worsen asthma severity. We previously demonstrated a detrimental link between PTSD-relevant fear behavior and allergen-induced lung inflammation associated with a mixed Th2/Th17-inflammatory profile in mice. However, the behavioral effects of Th2-skewed airway inflammation, typical to mild/moderate asthma, are unknown. Therefore, we investigated fear conditioning/extinction in allergen house dust mite (HDM)-exposed C57Bl/6 mice, a model of Th2-skewed allergic asthma. Behaviors relevant to panic, anxiety, and depression were also assessed. Furthermore, we investigated the accumulation of Th2/Th17-cytokine-expressing cells in lung and brain, and the neuronal activation marker, ΔFosB, in fear regulatory brain areas. HDM-exposed mice elicited lower freezing during fear extinction with no effects on acquisition and conditioned fear. No HDM effect on panic, anxiety or depression-relevant behaviors was observed. While HDM evoked a Th2-skewed immune response in lung tissue, no significant alterations in brain Th cell subsets were observed. Significantly reduced ΔFosB+ cells in the basolateral amygdala of HDM mice were observed post extinction. Our data indicate that allergen-driven Th2-skewed responses may induce fear extinction promoting effects, highlighting beneficial interactions of Th2-associated immune mediators with fear regulatory circuits.

Introduction

Chronic inflammation associated with immune diseases can predispose to multiple conditions including cancer, cardiovascular disease, and stroke (Greten and Grivennikov, 2019; Alfaddagh et al., 2020; Parikh et al., 2020). Recently, there has been a growing interest in studying the impact of chronic inflammation, particularly in the lung, on the brain and behavior (Bolton et al., 2013; Ray et al., 2020). One chronic inflammatory disease of the lung is asthma, a highly prevalent condition that impacts more than 300 million people worldwide (Asher and Pearce, 2014). Notably, asthma is considered a disease of childhood as ~11% of US children aged 15-19 are diagnosed with asthma making them vulnerable to other physical illnesses, including compromised mental and emotional health, into adulthood (Bui et al., 2021).

In this regard, strong evidence supports an association of asthma with psychiatric illnesses, particularly PTSD (for review see (Allgire et al., 2021)). A meta-analysis of GWAS studies identified a significant genetic correlation between asthma and PTSD (Nievergelt et al., 2019). Notably, the asthma-PTSD correlation was stronger than that observed between PTSD and systemic conditions such as cardiovascular disease, or metabolic disorders and was comparable to the association of PTSD with other psychiatric conditions such as depression. A correlation was not observed between asthma and major depressive disorder (MDD), schizophrenia (SCZ), bipolar disorder (BPD), or attention deficit/hyperactivity disorder (ADHD) suggesting potential selectivity for asthma-PTSD associations. Interestingly, among inner city children, PTSD symptoms were significantly associated with asthma severity (Vanderbilt et al., 2008), and a recent study reported higher prevalence of PTSD (50% of sample) in individuals with moderate to severe asthma compared to other psychiatric conditions, including depression and anxiety disorders (Paquet et al., 2019). Collectively, these lines of evidence strongly support shared contributory mechanisms between PTSD and asthma, in particular severe asthma. However, this association is not well understood as suitable preclinical models capturing the heterogeneity of asthma phenotypes are not available for mechanistic exploration.

Asthma is associated with a well-defined, progressive activation of both innate and adaptive immune responses to normally innocuous environmental antigens (allergens) (for review see (Lambrecht et al., 2019)). The heterogeneity in asthma severity (i.e. mild-moderate vs. severe asthma) stems from the development of unique inflammatory profiles. Traditionally, asthma has been characterized as a disorder of T helper 2 (Th2) cells as the Th2 cytokines IL-4, IL-5, and IL-13 are sufficient to drive asthma symptomology (Barlow et al., 2012; Foster et al., 2017; Peebles and Aronica, 2019). Recently, it has been suggested that more severe forms of asthma are associated with an immune response characterized by expansion of both Th2 cells associated with mild/moderate disease, as well as the expansion of Th17 cells, which produce cytokines like IL-17A or IL-17F (Irvin et al., 2014; Zheng et al., 2021). Murine asthma models characterized by allergen-driven expansion of predominantly Th2 cells, or co-expansion of Th2 and Th17 cells have been developed (Wakashin et al., 2008; Lajoie et al., 2010; Kim et al., 2019) and support clinical observations of an association between concomitant Th2/Th17 responses and more severe asthma (Irvin et al., 2014).

Utilizing a translationally relevant mouse model of severe allergic asthma (house dust mite allergen (HDM) administration in A/J mice) associated with a mixed Th2/Th17 response (Lajoie et al., 2010), we recently reported increased fear (freezing) during fear extinction, a PTSD-relevant behavior (but not anxiety, panic or depression-like behaviors) in allergen-treated A/J mice compared to allergen-naïve controls (Lewkowich et al., 2020). These changes were associated with an increased frequency of Th17 cells (but not Th2 cells) in the brain after allergen treatment. However, it remains to be investigated whether these effects are specific to Th2/Th17-driven severe airway inflammation since behavioral effects of Th2-skewed phenotypes were not studied. This is relevant for understanding whether the asthma-PTSD association is limited to severe airway inflammation or extends to mild-moderate asthma phenotypes as well.

Previous studies reported increased anxiety-like behavior in models of allergic rhinitis, allergic dermatitis, and allergic airway inflammation using ovalbumin (OVA) (Palermo-Neto and Guimarães, 2000; Tonelli et al., 2009; Zhuang et al., 2018; Dehdar et al., 2019; Salimi et al., 2019; Gholami-Mahtaj et al., 2022) or house dust mite (HDM) (Caulfield et al., 2017, 2018; Caulfield, 2021). However, to our knowledge, specific contribution of Th2-skewed airway inflammation on PTSD-relevant behaviors has not been investigated.

Given these considerations, the current study assessed the impact of HDM-induced Th2-skewed airway inflammation in C57Bl/6 mice (Gueders et al., 2009; Lajoie et al., 2010) on PTSD-relevant fear behavior and immunological T cell frequency in the lung and brain. Based on evidence supporting an association between PTSD and severe asthma, and our previous study showing increased frequency of brain Th17/IL17A+ cells with Th2/Th17 phenotype (Lewkowich et al., 2020), we hypothesized that Th2 only-skewed airway inflammation would not impact fear extinction or alter T cell frequency in the brain. To confirm a functional asthma phenotype, we measured airway resistance via a dose-response of methacholine, a muscarinic receptor agonist that induces airway smooth muscle constriction. To characterize the immune response in the lungs and brain, we assessed inflammatory cells recruited to the airways, cytokine production by immune cells in lung parenchyma, and conducted flow cytometric analysis of Th2- and Th17-relevant transcription factors GATA3 and Rorγt in lung and whole brain tissue. Assessment of behaviors relevant to panic, anxiety, and depression was also undertaken. To investigate recruitment of contributory brain areas, we conducted ΔFosB immunostaining, a surrogate marker for neuronal activation.

Methods

Animals

Male C57Bl/6 mice (cohort 1 n=24; cohort 2 n=16) were obtained from Jackson Laboratory at 7 weeks (Bar Harbor, Maine, USA). For consistency with our previous study on the Th2/Th17 model (Lewkowich et al., 2020) all measurements for the current study were performed in male animals. Mice were group housed 4/cage in a vivarium facility set to 23±4°C, humidity 30±6%, 14/10 h light-dark cycle (6am-8pm) and accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Food and water access was provided ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children’s Hospital Medical Center (CCHMC) and University of Cincinnati. Behavioral testing was conducted at 10-14 weeks of age between 8 am and 2 pm. Two cohorts were used: Cohort 1 underwent behavioral testing for exploratory and defensive behaviors relevant to panic, anxiety, depression, and fear one week following HDM/PBS treatment (see Fig. 1). For assessing HDM-induced alterations in inflammatory mediators in brain and lungs, a second behavior-naïve cohort (Cohort 2, Fig 1) was exposed to HDM/PBS. At 72 h following the last administration of HDM when peak lung inflammation has been reported (Lewkowich et al., 2008), we collected bronchoalveolar lavage (BAL), lung and brain tissue for flow cytometry and conducted airway hyperresponsiveness measurements. Separate groups of mice were used for airway hyperresponsiveness and measurement of brain inflammatory mediators.

Figure 1:

Timeline representing allergen treatment and behavioral paradigm. Mice received 3 intratracheal treatments of either house dust mites (HDM) or PBS separated by a week. (A) For cohort 1, animals were given a week of recovery post allergen treatment. Animals then underwent behavioral testing on the elevated zero maze (EZM), carbon dioxide (CO₂) inhalation context conditioning paradigm, forced swim test (FST), and contextual fear conditioning (numbers indicate days of testing). Tissue was collected 24h after the final day of behavioral testing for immunohistochemistry. (B) For cohort 2, mice received intratracheal HDM administrations like Cohort 1. At 72 h following the last treatment, mice were tested for methacholine airway resistance. Tissue was collected for assessment of brain mononuclear cells, inflammatory mediators in lung tissue and bronchoalveolar lavage fluid.

HDM Administration

As reported in our previous study (Lewkowich et al., 2020), following acclimation to the colony, mice were given 40 μl of 200 μg of aeroallergen House Dust Mite (HDM) (strain D. pteronyssinus, Product # XPB91D3A25, lot # 315580, Greer Labs, Lenoir, NC) or phosphate buffered saline (PBS) of the same volume intratracheally while anesthetized with isoflurane. HDM treatments were administered three times, each a week apart.

Behavioral Testing

Behavioral testing was conducted at 7 days after final HDM/PBS exposure (see Fig 1), for consistency with our previous studies with the Th2/Th17 severe asthma model (Lewkowich et al., 2020). Mice were tested on the elevated zero maze (EZM), carbon dioxide exposure (CO₂), and forced swim test (FST) to assess exploratory, defensive and learned helplessness behaviors relevant to anxiety, panic and depression, followed by contextual fear conditioning and extinction relevant to PTSD. The behavior battery was conducted in order of least to most stressful and the layout was selected based on evidence that anxiety- and exploration-related behaviors are impacted by prior assessment, while fear-associated behaviors are not significantly altered (Võikar et al., 2004). This timeline is also consistent with paradigms that simulate delayed manifestation of PTSD-relevant behaviors in the same animal.

Elevated Zero Maze

EZM was conducted as previously described by our group (Schubert et al., 2018; Lewkowich et al., 2020). The maze (Stoelting Co., Wood Dale, IL) consisted of four quadrants on a raised circle. Opposing quadrants consisted either of 1 cm high clear acrylic curbs or black acrylic walls 20 cm in height, characterized as the open and closed quadrants, respectively. Behavior was conducted at dim halogen lighting (24 lx on the open quadrant). Mice were initially placed in the closed quadrant for 5 minutes of video-recorded exploration. Between mice, the apparatus was cleaned with 10% EtOH. TopScan software (CleverSys. Inc., Reston, VA) scored time in open and closed quadrants, latency to enter open quadrant, and total distance traveled.

CO₂ inhalation paradigm

CO₂ inhalation is a translationally relevant interoceptive stimulus, that produces intense fear and panic attacks in individuals with panic disorder (Papp et al., 1993; Rassovsky and Kushner, 2003). CO₂ evoked fear (freezing) in rodents has been used by us and others. (Ziemann et al., 2009; Leibold et al., 2016; Vollmer et al., 2016; McMurray et al., 2020) as a panic-related behavioral assessment. Animals were placed in a dual vertical Plexiglas chamber (25.5 cm x 29 cm x 28 cm per chamber) and allowed to habituate for 7 minutes. The next day, animals were again placed in the chamber while CO₂ (10%; custom industrial mix in breathing air, Wright Brothers Inc., Cincinnati, OH) filled the upper chamber at an infusion rate of 10L/min. CO₂ concentration in the lower chamber was measured by CARBOCAP^® GM70 carbon dioxide meter (GMP221 probe with accuracy specification ± 0.5%) Vaisala, Helsinki, Finland. Ambient CO₂ concentration was confirmed to be 8-10 ± 0.5%. Mice were left in the lower chamber with CO₂ for 10 minutes. On the final day, the animals were placed in the lower chamber in the absence of CO₂ for five minutes to measure context conditioned freezing. On all days, freezing (the lack of all movement except respiration), was measured as a fear-relevant output utilizing FreezeScan software (CleverSys Inc., Reston, VA).

Forced Swim Test

The forced swim test was conducted as previously described (McMurray et al., 2019; Lewkowich et al., 2020). Mice were placed in a glass cylinder (14 cm diameter, 19 cm high) with 14 cm of water (24 ± 0.5 °C) and recorded for 6 minutes. Recordings were assessed for immobility (no active movement or floating without struggle) by a blind observer. Total immobility for minutes 2-6, as well as latency to immobility, was summed for individual animals and averaged within treatment group.

Contextual Fear Conditioning

A contextual fear conditioning paradigm was used (see Fig 5 layout). Fear acquisition, conditioned fear and extinction was investigated as we previously reported (McMurray et al., 2020) with modifications. Operant chambers housed in sound attenuated isolation cabinets were used (San Diego Instruments). The chamber floors consisted of stainless-steel grid bars that delivered scrambled electric shocks. The grid, floor trays and chamber walls were wiped with 10% ethanol and allowed to dry completely between animals. Mice were acclimated to the chamber for 5 min, then received 3 shocks of 0.5 mA intensity, 1s duration administered 1 min apart. The animals were placed in the chamber the next 6 days and recorded for 5 min without shocks to measure conditioned fear (day 2) and extinction (days 3-8). Freezing, defined as complete lack of movement except respiration, was measured using the Freeze Scan software (Clever Sys Inc.). We also assessed rearing behaviors on during extinction testing as a measure of an active defensive behavior. Frequency of rearing was scored by a trained observer blinded to experimental condition.

Figure 5:

Exposure to house dust mite induces extinction-specific effects on freezing without impacting fear acquisition or conditioned fear. Panel A shows the temporal layout of the contextual fear conditioning paradigm indicating exposure to foot shocks during acquisition (Acq), context exposure for conditioned fear (CF) and repeated context exposure for extinction. There were no significant group differences in freezing prior to foot shocks (baseline B) or post foot shocks (Shock) during acquisition (B) or following context exposure 24 h post acquisition for conditioned fear, CF (C). Mice previously treated with HDM showed significantly reduced freezing versus PBS when compared between CF and final extinction day, E7 (D) or as mean extinction freezing (E, left panel). No significant differences were observed in rearing behavior, a measure of active context exploratory behavior and motivation for escape (E, right panel). * p<0.05 versus baseline (panel B); versus PBS (panels D, E). Data are mean ± SEM; n = 12/group.

Airway hyperresponsiveness measurements

The flexiVent system (SCIREQ Scientific Respirator Equipment, Inc, Montreal, Quebec, Canada) was utilized to evaluate airway hyperresponsiveness 72 hours after final HDM or PBS exposure. Mice were anesthetized with a mixture of ketamine (90-120 mg/kg), xylazine (10-20 mg/kg) and paralyzed with pancuronium bromide (0.8-1.2 mg/kg). Tracheas were cannulated with an 18-gauge blunt cannula. Mice were ventilated at 150 breaths/min, 3.0 cm water positive and expiratory pressure, and allowed to stabilize on the machine for 2 minutes. Mice were then exposed to methacholine (0, 6.25, 12.5 and 25 mg/ml) aerosolized in PBS for 15 seconds and ventilated for an additional 10 seconds. Ventilation cycle measurements were taken until resistance peaked. Airways were then re-recruited by deep inflation and the next methacholine dose was administered.

Tissue preparation and flow cytometry

Tissue for flow cytometric analyses was collected at 72 h after the final PBS/HDM administration (Fig. 1, Cohort 2). This time point was selected as it represents the peak inflammatory response in the lung (Lewkowich et al., 2008). As an initial assessment and for consistency with our previous study (Lewkowich et al., 2020) we opted for whole brain analysis of mononuclear cells to identify broad changes in immune composition that may have occurred.

i). Lung tissue:

Lungs were removed, minced and placed in 6 ml of RPMI 1640 containing Liberase CI (0.5 mg/ml) (Roche Diagnostics) and DNase I (0.5 mg/ml) (Sigma) at 37 °C for 45 min. The remaining tissue was forced through a 70-μm cell strainer, and red blood cells were lysed with ACK lysis buffer (Thermo Fisher Scientific). Cells were washed with RPMI containing 10% FBS, viable cells were counted via trypan blue exclusion. Collected cells were plated at a density of 250,000 live cells per well in 96-well plates and restimulated in quadruplicate with either complete RPMI medium (RPMI media, 10% FBS, 0.1% β-mercaptoethanol, Penicillin, Streptomycin, L-glutamine), 30μg/ml HDM, or 5μg/ml Concanavalin A (Sigma) for 72 hours. Cell-free supernatants were collected and analyzed for cytokines via ELISA using antibodies from eBioscience: IL-5 (clones TRFK5, TRFK4), IL-13 (clones eBio13A, eBio1316H), IL17A (clones eBio17CK15A5, eBio17B7).

ii). Brain tissue:

Mononuclear cells were isolated from whole brain as previously described (Frank et al., 2006; Pino and Cardona, 2011) with modifications. Briefly, mice were anesthetized and perfused transcardially with ice-cold 1X HBSS. Brains were isolated and homogenized in phosphate buffered saline (PBS). Resulting homogenates were centrifuged (600×g for 6 min), supernatants were removed, and cell pellets were resuspended in 70% isotonic Percoll (GE Healthcare) at room temperature. This cell suspension was layered over a discontinuous 70%-30% Percoll density gradient and centrifuged for 30 min at 500×g. Cells were washed and then resuspended in sterile RPMI till further processing.

iii). Flow cytometry:

Single cell suspensions of lung and brain tissue were first incubated with Fc Block (mAb clone 2.4G2) to prevent non-specific Ab binding for 15 min. Following this, cells were incubated with fluorochrome-labeled antibodies to surface markers and incubated with a fixable Live-Dead Dye to exclude dead cells (Thermo Fisher Scientific). Cells were then fixed and permeabilized for 1 h using Foxp3 staining kit (Thermo Fisher Scientific). Intracellular Fc receptors were blocked using Fc Block suspended in Permeabilization Buffer (Thermo Fisher Scientific), followed by staining with cytokine- or transcription factor-specific mAbs suspended in Permeabilization Buffer. Data were acquired with an LSR-Fortessa flow cytometer equipped with lasers tuned to 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm, and digital DiVa Software. Spectral overlap was compensated, and data was analyzed using FlowJo software (Treestar Inc., Ashland, OR). All staining reagents used were purchased from Thermo Fisher Scientific, unless otherwise indicated. Clones used were as follows: BV605-conjugated anti-mouse CD90.2 (clone 53–2.1 (BioLegend)), APC-Cy7-conjugated anti-mouse TCRβ (clone H57-597), AlexaFluor700-conjugated anti-mouse CD3ε (clone 17A2), Brilliant Violet 711-conjugated anti-mouse CD4 (clone RM4-5 (BioLegend)); PE-eFluor-610-conjugated anti-mouse CD44 (clone IM7), Fixable Live/Dead e506 (Thermo Fisher Scientific), PE-conjugated anti-mouse Rorγt (clone AFKJS-9), eFluor 660 conjugated anti-human/mouse GATA3 (clone TWAJ).

iv). Bronchoalveolar lavage:

A cannula was inserted through the trachea and lungs were washed 3 times with 1ml Hank’s Balanced Salt Solution (HBSS, Gibco 14025076) to flush airways. Collected fluid and cells were placed into a 1.5ml centrifuge tube and centrifuged at 300xg for 6 minutes. Fluid was removed (stored at −80C) and the cell pellet was resuspended in 150ul ACK lysis buffer (Thermo Fisher Scientific) to lyse red blood cells and incubated at room temperature for 4 minutes. 500ul of PBS with 10% FBS was added to the cells to neutralize ACK and then the tubes were spun at 300xg for 6 minutes. Supernatant was aspirated, cells were resuspended in 500ul PBS with 10% FBS and kept on ice during total cell counting with a Hemacytometer. Approximately 50,000 cells were put onto a slide using a cytospin and then differentially stained using the DiffQuik stain kit (Camco, product # 702). Cells were then differentially enumerated as either monocytes/macrophages, eosinophils, neutrophils, lymphocytes, or epithelial cells.

Immunohistochemistry

Mice were transcranially perfused with 4% paraformaldehyde 24 hours after the final day of behavior for ΔFosB measurement, an indicator of prolonged neuronal activation. Brains were postfixed and placed in 30% sucrose until slicing. Brains were sliced on a sliding microtome at 30 μm and slices were stored in cryoprotectant (0.1 M phosphate buffer, 30% sucrose, 1% polyvinylpyrrolidone, and 30% ethylene glycol) at −20 °C. For immunohistochemistry, slices were transferred to 50 mM PBS (pH 7.4; 40 mM potassium phosphate dibasic, 10 mM potassium phosphate monobasic, and 0.9% sodium chloride) for 5 x 5 min rinses. Sections were then washed in 0.3% H₂O₂ in PBS for 10 minutes. Slices were again rinsed 5 x 5 minutes followed by blocking for 1 h at RT (50 mM PBS, 0.5% bovine serum albumin (BSA), and 0.4% Triton X-100). Slices were then incubated with anti-ΔFosB primary antibody (1:5000 Santa Cruz Biotechnology, sc-7203, Santa Cruz, CA) in blocking solution overnight at 4 °C. On day 2, sections were rinsed 5 x 5 minutes and incubated in secondary antibody (1:400 biotinylated anti-rabbit, Vector Laboratories, Inc., Burlingame, CA) for 1 h followed by another set of rinses. Slices were then placed in avidin-biotin complex using ABC Vectastain kit (1:800 A and B) for 1h. After another set of washes, sections were incubated in diaminobenzidine (DAB, Pierce, Rockford, IL) for 10 minutes. The sections were then rinsed in PBS and mounted onto microscope slide following dehydration and xylene clearance. Slides were cover slipped using DPX mounting medium (Sigma, 44581).

Imaging

ΔFosB stained sections were imaged using AxioImager ZI microscope (Zeiss) with apotome (z-stack) capability (Axiocam MRM camera and AxioVision Release 4.6 software; Zeiss). Processing was performed as previously described (Lewkowich et al., 2020; McMurray et al., 2020). Areas were selected based on their role in stress, fear, and emotional regulation, including the amygdala, medial prefrontal cortex, hippocampal dentate gyrus, bed nucleus of stria terminalis, and paraventricular nucleus of the hypothalamus, and were identified utilizing the mouse brain atlas of Franklin and Paxinos (Franklin K; Paxinos, 2008). Each region had at least 4 images per mouse at similar distance from bregma across animals. ΔFosB+ cells were quantified with the Image-J “cell counter” tool by an investigator blind to the experimental group. Quantities were reported via averaging cell counts of each section within animal then averaged within groups to determine group means.

Data Analysis and Statistics

Figures represent data as mean ± SEM. Analyzed data were tested for normality and met assumptions for tests applied. For fear conditioning acquisition and extinction as well as CO₂ inhalation, a two-way repeated measures ANOVA was used with treatment and time as variables. Where main effects occurred, Sidak’s post hoc was applied. EZM, FST, and ΔFosB counts were analyzed using a two-tailed student’s t-test. Statistical significance was set to p < 0.05. All analyses were conducted with Prism 8.0 (GraphPad Software, Inc., La Jolla, CA).

Results

HDM exposure in C57Bl/6 mice generates a Th2-skewed response in the lung but no alterations in T cell frequency in the brain

72h after the last of 3 HDM treatments (a time point that associates with peak inflammatory response in the lungs (Lewkowich et al., 2008)), the asthmatic response induced in C57BL/6 mice was assessed. As expected HDM-exposure was sufficient to induce modest airway hyperresponsiveness (AHR) (Fig 2A), as indicated by significantly higher resistance to methacholine in HDM mice versus PBS (2-way RM ANOVA, F _{(4, 56)} = 4.17, p = 0.005). To assess the pulmonary inflammatory response, we assessed cellular infiltration into the airways by performing a bronchoalveolar lavage and collecting cells from the fluid (BALF). HDM exposure was associated with robust accumulation of inflammatory cells in the airways (t test, t₁₆ = 7.049, p<0.0001) (Fig 2B). Differential cell counts performed on these BALF cells demonstrated that while monocytes/macrophages predominated in the lungs of PBS-exposed animals (~95% of cells), in the HDM-exposed animals the predominant cell type present was eosinophils (~60%) (t test, t₁₆=11.65, p<0.0001 vs PBS) (Fig 2C). Of the remaining cells, ~25% were monocytes/macrophage, whereas neutrophils and lymphocytes represented <5% of the total cells each, albeit still significantly elevated in the HDM-exposed animals (t test neutrophils: t₁₆=2.994, p=0.009 vs PBS; lymphocytes: t₁₆=4.461, p=0.0005) (Fig 2C). These observations are consistent with a predominantly Th2/biased immune response in the lungs.

Figure 2:

House dust mite exposure in C57BI/6 mice evokes airway hyperresponsiveness and generates a Th2-skewed response in the lung. (A) HDM-treated animals demonstrated increased airway hyperresponsiveness (AHR) as indicated by significantly higher resistance to methacholine. (B) HDM exposure was also associated with an increase in inflammatory cells present in the bronchoalveolar lavage fluid (BALF) (B). (C) Differential cell counts on BALF inflammatory cells revealed that eosinophils represent the predominant cell population in HDM exposed mice. Comparatively minor (albeit significant) increases were also observed in neutrophils and lymphocytes. (D) Flow cytometric analysis in lung tissue revealed a marked increase in the frequency of GATA3+ cells in HDM mice (left panel), and a shift from innate (ILCs, γδ T cells) to adaptive GATA3+ cells (CD4+ T cells) (right panel). (E) Rorγt-expressing cells were also increased upon HDM challenge (left panel), although RORγt-expressing cells did not show a marked shift in distribution from innate immune cells (ILCs, γδ T cells) to CD4+ cells (right panel). (F) HDM-induced re-stimulation of T cell cytokine production in vitro using whole lung cell suspensions from mice previously treated with PBS and HDM revealed significantly increased production of IL-5 and IL-13 in the HDM group, while IL-17A was not significantly different. * p<0.05 versus PBS. Data represented as mean ± SEM (n=8-10/group).

To further characterize the immune response, lung mononuclear cells were extracted for flow cytometric analysis. To characterize the nature of infiltrating lymphocytes, we measured the frequency of cells expressing the Th2-relevant transcription factor, GATA3, and Th17-relevant transcription factor, Rorγt. Our flow cytometric panel categorized further GATA3- and Rorγt-producing cells as well as γδ T cells (CD90.2⁺, CD3⁺, TCRβ^neg), CD4+ T Cells (CD90.2⁺, CD3⁺, TCRβ⁺, CD4⁺), CD8+ T cells (CD90.2⁺, CD3⁺, TCRβ⁺, CD4^neg), and innate lymphoid cells (ILCs) (CD90.2⁺, CD3^neg, TCRβ^neg) (see Supplementary Fig. 1 for gating strategy). Consistent with inflammatory cell data, HDM exposure almost tripled the recruitment of GATA3+ cells into the lungs (t test, t₁₄ = 4.823; p = 0.0003). (Fig 2D). In PBS-exposed animals, ILCs represented the majority (~75%) of GATA3+ cells, whereas CD4+ T cells accounted for ~20% of GATA3+ cells (Fig 2D, right panel). Following HDM exposure, this ratio flipped, with ~85% of GATA3+ cells also staining for CD4, and ~10% of GATA3 cells lacking lineage specific markers (i.e. ILCs) (2-way ANOVA, F _{(3, 57)} = 340.44, p < 0.0001) (Fig 2D, right panel). In contrast to the robust recruitment of CD4+ cells producing Th2-associated cytokines, increases in Rorγt-expressing cells were more limited (~2 fold increase), albeit still statistically significant (t test, t₁₄ = 4.370, p = 0.006). (Fig 2E). Importantly, the distribution of cells expressing Rorγt was also markedly different. In PBS exposed animals, ~70% of cells were innate γδ T cells, and 20% of cells were CD4+ T cells, whereas in HDM-exposed animals γδ T cells and CD4+ T cells represented 40% and 50% of all Rorγt-expressing cells, respectively (2-way ANOVA, F _{(3, 60)} = 25.38, p < 0.0001) (Fig 2E, right panel).

Finally, as flow cytometric analysis reflects the total capacity to produce T cell cytokines, regardless of T cell specificity, we also assessed the antigen-specific stimulation of T cell cytokine production using whole lung cell suspensions restimulated in vitro with HDM allergen (Fig 2F). Consistent with the eosinophil dominated inflammatory response, total lung cells from HDM-exposed C57Bl/6 mice produced significantly more IL-5 (t test, t₁₆= 3.824, p = 0.0015) and IL-13 (t test, t₁₆ = 3.931, p = 0.0012) in response to HDM restimulation than cells from PBS-exposed mice, while production of IL-17A was not significantly elevated (t test, t₁₆ = 1.535, p = 0.1443) (Fig 2F).

As we had previously observed a selective increase in the frequency of Rorγt-expressing cells in the brains of HDM-exposed A/J mice in a model of severe asthma (Lewkowich et al., 2020), we next examined the frequency of GATA3- or Rorγt-expressing cells in the brains of HDM-exposed C57Bl/6 animals. In allergen-exposed C57Bl/6 mice, HDM-exposure did not alter the overall frequency of GATA3-expressing cells (Fig. 3A) (t test, t₁₃ = 0.387, p = 0.705). In the brain, approximately half of the GATA3+ cells were γδ T cells, with CD4+ T cells and ILCs making up the remaining proportions as there was very low expression of GATA3 in CD8+ T cells (Fig. 3B). These proportions were not altered by HDM exposure (Fig 3B). In contrast to our previous data showing an increased frequency of Th17 cells in the brain in a model of severe asthma (Lewkowich et al., 2020), HDM did not alter the frequency of Rorγt-expressing cells in the current model of mild asthma (Fig. 3C) (t test, t₁₃ = 0.6677, p = 0.516). For Rorγt+ cells, ILCs were the primary Rorγt-expressing cells (~55%), followed by γδ T cells (~35%). CD4+ and CD8+ T cells produced a minimal amount of Rorγt (Fig. 3D). As with GATA3-expressing cells, HDM exposure did not change the overall proportions. Collectively, these data suggest that despite strong HDM-induced Th2 inflammation in the lungs of HDM-exposed C57Bl/6 mice, HDM-exposure does not alter lymphocyte populations in the brain.

Figure 3:

Pulmonary allergen exposure does not increase the frequency of asthma relevant T helper cells in the brain. (A) No significant differences in the frequency (A) or the relative proportions (B) of GATA3+ cells was observed in brain tissue of PBS and HDM groups. Similarly, HDM exposure was not associated with an altered frequency (C) or proportion (D) of Rorγt-expressing cells in the brain. Data are expressed as mean ± SEM; n=7-8/group.

HDM treatment does not impact anxiety-, panic- or depression-relevant behaviors.

To examine the broad impact of allergic inflammation on altering mouse behaviors such exploration, learned helplessness or defensive behaviors to a panic-relevant challenge, we compared performance of PBS- and HDM-exposed animals in EZM, FST, and CO₂ inhalation paradigms, respectively. No significant group differences were observed in time spent in the open arm of the EZM (t test, t₂₂ = 1.265; p = 0.05) (Fig. 4A). In the FST, there was no significant difference in total immobility time (t test, t₂₂ = 0.5539; p > 0.05) (Fig 4B) nor for the latency to immobility (t test, t₂₂ = 0.5445; p > 0.05) (Fig. 4C). Freezing was assessed in the CO₂ paradigm for spontaneous and conditioned defensive behaviors relevant to panic. A repeated measure 2-way ANOVA comparing freezing during habituation, CO₂ inhalation and context exposure revealed a main effect of time (2-way RM ANOVA, F _{(1.189, 26.16)} = 69.36; p <0.0001) but not treatment (2-way RM ANOVA, F _{(1, 22)} = 0.6713, p = 0.4214) or time x treatment interaction (2-way RM ANOVA, F _{(2, 44)} = 0.162, p = 0.8510), (see Fig 4D). Sidak’s multiple comparison test revealed that mice exhibited increased freezing upon CO₂ exposure and context re-exposure for conditioned fear compared to habituation, but prior HDM exposure did not impact this behavior as compared with the PBS control group.

Figure 4:

HDM exposure does not impact exploratory behavior, learned helplessness or CO₂-associated spontaneous or context conditioned freezing. (A) There was no significant difference between PBS and HDM groups in time spent in open area in elevated zero maze (EZM). In forced swim test (FST) there were no group differences in time spent immobile (B) or the latency to immobility (C). (D) Significantly higher freezing versus habituation was observed during CO₂ inhalation (CO₂) and context re-exposure 24 h following CO₂ for conditioned fear (CF). However, no significant differences in freezing were observed between PBS and HDM groups. * p<0.05 versus habituation (panel D). Data are mean ± SEM; n = 12/group.

Attenuated freezing during fear extinction in HDM treated mice with no effects on fear acquisition or conditioned fear.

PBS and HDM mice were tested in a contextual fear conditioning paradigm for assessing effects on fear acquisition, conditioned fear and extinction (Fig. 5A). No group difference in baseline freezing was observed during the habituation phase (Fig 5B). For acquisition (Fig. 5B), a 2-way RM ANOVA revealed a main effect of time (F _{(1, 22)} = 57.19;p< 0.0001) but not treatment (F_{(1, 22)} = 0.102; p = 0.8816) or time x treatment interaction (2-way RM ANOVA, F _{(1, 22)} = 0.1126; p = 0.7289) suggesting no effects of HDM-treatment on fear acquisition. Context exposure at 24-hours post acquisition revealed no significant group difference in freezing (t test, t₂₂ = 0.8144; p = 0.4241), suggesting that HDM-exposure does not affect consolidation of fear (Fig. 5C). Repeated exposure to context for extinction revealed lower freezing in HDM-exposed mice as compared with PBS mice (Fig. 5D). Comparison of freezing on conditioned fear (CF) and last day on extinction (E6) between PBS and HDM groups using 2-way RM-ANOVA revealed a significant main effect of treatment (F _{(1, 22)} = 10.60; p = 0.0142) and time (F _{(1, 22)} = 13.45; p = 0.0122), but no significant interaction (F _{(1, 22)} = 3.397; p = 0.1837), suggesting that both groups exhibited extinction, however the HDM-treated group had significantly lower freezing. Of note, although there was a significant effect of time on freezing across days, the reduction in freezing was modest as compared to a previous study by our group in this strain (Vollmer et al., 2013), potentially, an effect of treatment manipulations in this study. A comparison of mean extinction freezing (Fig. 5E) also revealed significantly lower freezing in HDM treated mice vs the PBS group (t test, t₂₂ = 2.494; p = 0.0206). We also assessed rearing frequency during extinction. Rearing has been reported to represent context exploratory behavior and escape motivation (Shansky, 2015; Biagioni et al., 2016) and was included in addition to freezing to assess whether reduced freezing in HDM mice was due to active defensive coping. No significant difference in rearing frequency was observed between HDM and PBS mice (t test, t₂₂ = 1.042, p = 0.309) (Fig. 5E), suggesting that lower freezing in HDM mice was not an outcome of a behavioral shift towards “active” defensive behaviors.

HDM alters neuronal activation in the basolateral amygdala, a fear regulatory region

ΔFosB staining was conducted in brain tissue collected post behavior to capture enduring changes in neuronal activation in our paradigm. As ΔFosB is a truncated version of FosB, a protein expressed in conditions of transcription and response to stimuli, it is longer-lasting than cFos and thus is an indicator of persisting neuronal activation. A regional analysis of several fear and stress-regulatory areas revealed significantly lower ΔFosB cell counts in the basolateral amygdala (BLA) of HDM-treated mice (t test, t₂₂ = 2.161, p = 0.0418) (Fig. 6A). Surprisingly, no significant group differences were observed in other regions such as the central amygdala (CeA) (t test, t₂₂ = 0.1534; p = 0.8795), medial prefrontal cortex (mPFC) (t test, t₂₂ = 0.2218; p = 0.8265), bed nucleus of the stria terminalis (BNST) (t test, t₂₂ = 0.9696, p = 0.3433), the hippocampal dentate gyrus (DG) (t test, t₂₂ = 1.304; p = 0.2065), or paraventricular nucleus (PVN) (t test, t₂₂ = 1.020; p = 0.3196) (Figs 6B-F). Our data suggests sub-region selective effects of HDM on neuronal activation within the amygdala.

Figure 6:

HDM exposed mice show altered neuronal activation. Significant decrease in delta FosB immunopositive (ΔFosB⁺) cells was observed in HDM versus PBS treated mice following behavior within the basolateral amygdala (BLA) (A). No significant difference was seen in central amygdala (CeA) (B), medial prefrontal cortex (mPFC) (C), bed nucleus of the stria terminalis (BNST) (D), dentate gyrus (DG) (E), and paraventricular nucleus (PVN) (F). * p<0.05 versus PBS. Data are mean ± SEM; n = 12/group.

Discussion

The current study indicates that intratracheal exposure to the aeroallergen HDM, a translationally relevant model of asthma associated airway inflammation, produces selective behavioral effects on fear extinction in a mouse strain that exhibits a Th2-skewed immune response in the lung. Our data also reveal reductions in persistent neuronal activation within the basolateral amygdala, a fear-regulatory region in allergen-exposed mice post behavior. Collectively, these observations suggest regulatory effects of asthma pertinent Th2 immune mediators on fear extinction behavior.

CD4+T helper (T_H) cells play crucial roles in choreographing adaptive immune responses, and the Th2 subset and associated cytokines are central to pathologic responses linked with asthma (Ray and Cohn, 1999; Lambrecht et al., 2019; Hammad and Lambrecht, 2021). The pathophysiology of type 2 inflammation is driven by both the innate immune system triggered by allergens involving type 2 innate lymphoid cells (ILC2) and the adaptive immune system, triggered by contact with an allergen that activates Th2 cells. Both ILC2 and Th2 cells produce the type-2 cytokines (interleukin (IL)-4, IL-5 and IL-13), each with several roles in the airway inflammation cascade (Lambrecht et al., 2019). Although Th2-driven responses are well understood in the context of airway inflammation, the impact of these responses on brain immune responses, or behavior is relatively unknown.

Significant strain differences exist in the magnitude of airway responsiveness and immune response to allergens, and we hypothesized that these differences may have effects on immune mediators produced locally within the brain. A comparison of airway hyperresponsiveness and airway remodeling in mouse strains systemically exposed to OVA followed by aerosol challenge (a common model for airway inflammation) reported the highest airway responsiveness and substantially greater inflammatory response (IL-4, IL-5, IL-13, IFN-γ, TGFβ) in A/J mice, whereas C57Bl/6 mice exhibited minimal airway remodeling and increases in IFN-γ (Shinagawa et al., 2003). Herein, we show that HDM treatment in C57Bl/6 mice evoked a Th2-skewed inflammatory response reflected by an increase in GATA3-expressing CD4+ T cells in the lungs, pronounced eosinophil recruitment to airways, and robust Th2-cytokine production in lung cell cultures. In contrast, while recruitment of Rorγt+ cells in the lungs was also observed, Rorγt-expressing cells were predominantly cells of the innate immune system (i.e. γδ T cells), there was little recruitment of pulmonary neutrophils after allergen challenge, and allergen-treated mice did not display significantly elevated IL-17A production in lung cell cultures. Taken together, these data suggest more Th2-biased immune responses associated with mild/moderate asthma in C57Bl/6 mice. Interestingly, flow cytometric assessment of whole brain mononuclear cells revealed no differences in the frequency of GATA3- or Rorγt-expressing cells between control or HDM-treated groups. This lack of HDM-induced Th cell immune responses in the brain is in contrast to our previous data in a Th2/Th17 airway inflammation model where HDM-exposed A/J mice elicited mixed Th2/Th17 recruitment in the lung and a significant increase in the frequency of IL-17A+ cells in the brain (Lewkowich et al., 2020). Collectively, previous, and current data suggest that C57Bl/6 mice represent a suitable model for allergen-induced increase in pulmonary Th2-associated mediators, however, no alterations in the frequency of brain Th cell-associated mediators is observed in this strain.

The role of peripheral proinflammatory mediators in regulating behavior and cognitive function has been shown previously, with both beneficial and detrimental effects, depending on the immune product involved. In this regard, a link between adaptive immunity and regulation of cognitive learning and memory processes has been reported with pro-cognitive effects of Th2 cells and detrimental impacts of Th17 cells (Brynskikh et al., 2008; Kipnis et al., 2012; Cipollini et al., 2019). Previous work from our group in HDM-exposed A/J mice that develop a Th2/Th17-skewed response showed compromised fear extinction without impacting fear conditioning (Lewkowich et al., 2020). Based on data in the mixed Th2/Th17 airway inflammation model, we anticipated either no change in extinction freezing (to support that fear extinction deficits are Th17/IL-17A associated) or increased freezing (supporting that Th2-associated mediators contribute to extinction deficits) in the current Th2-skewed model. However, contrary to these predictions, extinction freezing was significantly lower in the HDM treated group, suggesting beneficial effects of pulmonary Th2-associated immune mediators on extinction learning. These data are consistent with previous work reporting a positive regulatory influence of Th2 immune mediators on cognitive behaviors and memory (Derecki et al., 2010; Kipnis et al., 2012). Our data suggest that: a) extinction learning mechanisms may be selectively vulnerable to modulation by immune mediators generated by allergen-induced airway inflammation, and b) the nature of the inflammatory response (Th2 versus Th17) determines the directionality of the effect on fear extinction.

Currently, the link between pulmonary Th2 immune responses in C57Bl/6 mice and beneficial behavioral effects is not clear. Previous studies reported a distinct immune response in C57Bl/6 mice that promoted protection from allergen-induced effects compared to the other strains (Hirota et al., 2009; Azevedo et al., 2021). In an acute and chronic OVA sensitization model, C57Bl/6 mice developed a Th2 immune response to allergen but did not show activation of signals downstream of Th2 inflammation and did not develop structural changes in the airways when compared to BALB/c mice (Hirota et al., 2009). Furthermore, increased CD4+ CD25+ Foxp3+ Treg cells were observed in the bronchoalveolar lavage fluid (BALF) of OVA sensitized C57Bl/6 mice compared to A/J and BALB/c mice (Azevedo et al., 2021). It is probable that specific immune mediators generated in this strain are responsible for the improved fear extinction observed in our model. In support, multiple studies have reported beneficial modulatory effects of CD4+ CD25+ Foxp3+ Tregs on behavior (Kim et al., 2012; Liu et al., 2020), and Tregs were required for the fear-reducing effects of immunoregulatory microbial therapies (Reber et al., 2016). Recently, Th2-relevant cytokines, such as IL-4 and IL-13, have been found to act as neuromodulators on brain cells such as microglia, astrocytes, and even neurons (Herz et al., 2021). Our immune analysis of the brain was limited to the identification of IL-13, which does not rule out other Th2-relevant mediators such as IL-4. Mice deficient in IL-4 and IL-13 exhibit learning behavior deficiencies (Derecki et al., 2010; Brombacher et al., 2017). It would be important to assess alterations in other T cell subtypes, such as Tregs, and Th2-relevant cytokines in modulating extinction learning in the HDM-C57Bl/6 paradigm.

Extinction-selective effects in C57Bl/6 and A/J models of airway inflammation suggest a primary role for immune interactions within extinction-regulatory brain areas. Consistent with this, in the severe asthma model of HDM-exposure in the A/J strain, mice with increased extinction freezing elicited higher ΔFosB+ cell counts within the basolateral amygdala (BLA) and the prefrontal cortex (PFC) (Lewkowich et al., 2020). In contrast, HDM-treated C57Bl/6 mice with reduced extinction freezing elicited decreased BLA cell counts with no change in the PFC. These differences may be due to discrete immune mediators, regulatory mechanisms and cellular activation timelines involved these models. The BLA is a key extinction-regulatory site (Herry et al., 2006; Oitzl et al., 2012; Saha et al., 2022) and has reciprocal interactions with the medial prefrontal cortex, another area relevant to fear extinction (Little and Carter, 2013). An increase in BLA neuronal firing has been reported following peripheral immune challenges, such as lipopolysaccharide (LPS) and pro-inflammatory cytokines such as interleukin 1β (Munshi and Rosenkranz, 2018). Modulation of neuronal activity in the BLA by allergens is also supported by previous studies showing a significant increase of delta and theta low field potentials following intranasal OVA challenge (Dehdar et al., 2022). Interestingly, studies on individuals with asthma have reported an association of amygdala metabolism with airway inflammation (Rosenkranz et al., 2022). The exact mechanisms by which airway inflammation and T cell-associated immune mediators modulate BLA function in relation to fear extinction need to be investigated in future studies.

HDM treatment in C57Bl/6 mice had no effect on defensive behaviors relevant to anxiety, depression and panic. This concurs with our previous observations showing no effects of HDM exposure on these behaviors in A/J mice with Th2/Th17 expansion (Lewkowich et al., 2020), and with studies by Caulfield et al. (Caulfield et al., 2017, 2018, 2021) showing no effects of HDM-induced airway inflammation in BALB/c mice on anxiety-like behavior. In contrast, other models of airway inflammation, for example the OVA-sensitization-challenge and repeated tree pollen administration paradigms, have been reported to have significant effects on anxiety-relevant behaviors (Costa-Pinto et al., 2005; Tonelli et al., 2009). These differences may stem from the differential nature and intensity of antigens as well as timing and route of administration, as previous work suggests that the pathophysiologic pattern of asthma and associated inflammation may vary according to the mode of induction (Kim et al., 2019).

It would be important to discuss possible pathways that may translate airway immune responses into pro-extinction behavior. Our current study did not produce alterations in T cell-associated mediators in whole brain, however, regional differences, possibly at sites that interface between brain and blood, or cerebrospinal fluid may be present. Previous work has shown that peripheral T cell manipulations can regulate the frequency of CD4+ cells and Th2-related cytokine concentrations within CSF enriched compartments such as the meninges, choroid plexus, and areas lining the ventricles in mice (Derecki et al., 2010), and strong evidence supports a role of meningeal Th2 cells in regulating neuronal function and fear memory (Herz et al., 2021). Other BBB-compromised sites such as the circumventricular organs may also contribute given their role in mediating behavioral responses to physiological and immune challenges (Ferguson, 2014), T cell homing (Song et al., 2016), and neuronal projections to limbic sites regulating emotional behaviors (Swanson and Lind, 1986; McKinley et al., 2003).

The potential translational relevance of our findings to the asthma-PTSD association is unclear. Collective evidence from the current results and our previous report (Lewkowich et al., 2020) would indicate that the presence of a mixed Th2/Th17 immune response (as seen in more severe airway inflammation) is consistent with the observed extinction impairments in PTSD. On the contrary, immune mediators linked with a Th2-skewed response appear to have beneficial protective effects. It would be important to determine if individuals with asthma and PTSD express Th17-skewed immune mediators or increased Th2 mediators in individuals who do not develop PTSD following trauma. Interestingly, previous studies (Zhou et al., 2014; Busbee et al., 2022) report increased proportions of Th17 but unchanged Th2 CD4+ T cell subsets in PTSD patients, although the presence/absence of inflammatory disease specifically asthma was not reported.

Although our results provide information on the association of airway inflammation and regulation of fear, there are limitations and avenues for future investigation. The current study did not explore regional immune alterations, which would facilitate mechanistic investigations in future studies. It would be important to compare differential outcomes of airway inflammation between Th2 and Th2/Th17 phenotypes in different strains. In this regard, an interesting manipulation would be to shift HDM-induced Th cell responses to capture pro- or anti-extinction effects. For example, previous work from our group reported that HDM treatment in the presence of an anti-C5aR antibody induces a mixed Th2/Th17 phenotype and severe airway inflammation in an otherwise Th2-skewed strain (Lajoie et al., 2010). The behavioral implications of such manipulations need to be investigated. Lastly, our study was confined to male mice. Lastly, our study was confined to male mice. Given that females develop more robust airway hyperresponsiveness (Melgert et al., 2005; Richgels et al., 2017) and elicit higher fear responses (Maeng and Milad, 2015; Shansky, 2015), it would be important to study outcomes in female mice.

In conclusion, our study reveals that mild-moderate airway inflammation characterized by allergen-driven Th2 type expansion may induce fear extinction promoting effects, highlighting beneficial interactions of Th2-associated immune mediators with fear regulatory circuits.

Supplementary Material

1

Supplementary Fig 1: Gating strategy to classify types of cytokine producing cells. Cells were stained as described in the Materials and Methods second. (A) Initial gating on the basis of size was used to discriminate cells from debris (left panel), live cells (middle panel) and lymphocyte sized cells (right panel). Subsequent gating in HDM-exposed (B) and PBS-exposed (C) animals allowed the identification of cytokine producing cells. In both cases cells were first gated based on the expression of GATA3, or Rorγt. Subsequent gating allowed the identification of αβ T cells (CD3⁺TCRβ⁺), γδ T cells (CD3⁺TCRβ^neg) and Innate Lymphoid Cells (ILCs – CD3^negTCRβ^neg). Gated αβ T cells were subsequently classified as CD4⁺ or CD8⁺ (CD4^neg). Lung cell data shown. A similar gating strategy was used to identify cytokine producing cells in the brain.

Highlights.

• House dust mite (HDM) induces a Th2-skewed lung immune response in C57BI/6 mice.

• HDM treated mice elicit lower freezing during fear extinction.

• No significant alterations in brain Th cell subsets were observed in HDM mice.

• HDM-exposed mice had reduced ΔFosB cells in the basolateral amygdala.

• Data highlight beneficial interactions of Th2-immune mediators with fear circuits.