Peri-adolescent asthma symptoms cause adult anxiety-related behavior and neurobiological processes in mice

Abstract

Human and animal studies have shown that physical challenges and stressors during adolescence can have significant influences on behavioral and neurobiological development associated with internalizing disorders such as anxiety and depression. Given the prevalence of asthma during adolescence and increased rates of internalizing disorders in humans with asthma, we used a mouse model to test if and which symptoms of adolescent allergic asthma (airway inflammation or labored breathing) cause adult anxiety- and depression-related behavior and brain function. To mimic symptoms of allergic asthma in young BALB/cJ mice (postnatal days [P] 7-57; N=98), we induced lung inflammation with repeated intranasal administration of house dust mite extract (most common aeroallergen for humans) and bronchoconstriction with aerosolized methacholine (non-selective muscarinic receptor agonist). Three experimental groups, in addition to a control group, included: (1) "Airway inflammation only", allergen exposure 3 times/week, (2) "Labored breathing only", methacholine exposure once/week, and (3) "Airway inflammation+Labored breathing", allergen and methacholine exposure. Compared to controls, mice that experienced methacholine-induced labored breathing during adolescence displayed a ∼20% decrease in time on open arms of the elevated plus maze in early adulthood (P60), a ∼30% decrease in brainstem serotonin transporter (SERT) mRNA expression and a ∼50% increase in hippocampal serotonin receptor 1a (5Htr1a) and corticotropin releasing hormone receptor 1 (Crhr1) expression in adulthood (P75). This is the first evidence that experimentally-induced clinical symptoms of adolescent asthma alter adult anxiety-related behavior and brain function several weeks after completion of asthma manipulations.

Keywords: Adolescence; Anxiety; Asthma; House dust mite; Methacholine; Sex differences.

Figure 1. Study design and timelines

(A) Study design with four experimental conditions to manipulate peri-adolescent asthma symptoms to determine adult behavioral and physiological outcomes. Each experimental/control condition received a control or experimental treatment from each category (airway inflammation and labored breathing). (B) Timeline for peri-adolescent experimental manipulations. Dashed arrows indicate house dust mite (HDM) treatments to stimulate lung inflammation. Solid arrows indicate methacholine (MCH) treatments to induce labored breathing. HDM began earlier than MCH because significant inflammation requires several weeks of HDM exposure [74].

Figure 2. Peri-adolescent labored breathing and enhanced pause (Penh) values

Top: The mean number of labored breathing events during weekly methacholine (MCH) or vehicle exposure presented for the four treatment groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Mice that experienced MCH (LB and AI+LB) had significantly higher scores than those that did not experience MCH (CON and AI), indicated by *** (p<0.001). Bottom: MCH treatments also caused significantly higher mean enhanced pause (Penh) values compared to saline on P36, 43, 50, and 57, indicated by * (p<0.05).

Figure 3. Late adolescent fecal corticoid concentrations

Mean fecal corticoid metabolite concentrations in late adolescent (P59) male and female mice in the four experimental groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Female mice had significantly greater fecal corticosteroid metabolite concentrations during the diurnal trough compared to males, indicated by *** (p<0.001). Left side: Male mice that experienced MCH (LB and AI+LB groups) had a trend toward decreased fecal corticoid concentrations compared to males that did not experience MCH (CON and AI groups), indicated by # (p<0.10). Right side: The reverse was true for female mice, with a trend toward increased fecal corticoid concentrations in MCH (LB and AI+LB) vs. non-MCH (CON and AI) mice, indicated by # (p<0.10). Bars indicate means with standard error. [Contrasts & pairwise group comparisons: For females, there was a trend for increased fecal corticoids in AI+LB vs. CON females (p<0.10) with no other group differences identified in pairwise comparisons. For males, there were no differences between individual groups.]

Figure 4. Adult behavior

Behavioral measures for the following four treatment groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Adult mouse (P60) behavior in elevated plus maze (EPM) (A–C) indicates that adolescent MCH caused increased adult anxiety-live behavior but not locomotion: there was an LB main effect on time on open arms (A) and entries into open arms (B), but not on total entries into open and closed arms (C), main effect of LB indicated by * (p<0.05). No significant differences were observed in percent sucrose consumed by adult mice (P66) in sucrose preference test (SPT) (D). Bars indicate means with standard error. [Contrasts & pairwise group comparisons: LB and AI+LB groups spent less time on open arms of the EPM than the CON group (p<0.01 and 0.05); pairwise comparisons indicated no other differences among groups.]

Figure 5. Adult lung state and cytokine expression

Lung function measures for the four treatment groups: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Adolescent HDM treatments (AI and AI+LB groups) led to increased mucus (A), average inflammation patch length (B), number of inflammation patches (C), and gene expression (mRNA) for IL-4 (E), IL-5 (F), and IL-13 (G). Mucus and IL-5 expression were both greater in females compared to males. Collagen measures were not significantly different across groups (D). Main effects of AI and Sex indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001). [Contrasts & pairwise group comparisons: AI and AI+LB groups had significantly more mucus (p<0.001) and more inflammation (length and number of patches, p<0.001) than both CON and LB groups. Compared to the CON group, AI had more gene expression of IL-4 (p<0.05), and AI and AI+LB groups had more expression of IL-5 (p<0.01 and 0.05), and IL-13 (p<0.001). Further pairwise comparisons indicated that the AI group had greater expression of IL-5 (p<0.01) and IL-13 (p<0.001) than the LB group, and the AI+LB group had greater IL-13 expression than the LB group (p<0.05).]

Figure 6. Adult basal corticosterone and cytokine concentrations in serum, and transporter/receptor gene expression in the brain

The four experimental groups include: Control (CON), Airway Inflammation (AI), Labored Breathing (LB), and Airway Inflammation + Labored Breathing (AI+LB). Trending effect of AI indicated by # (p<0.10) for mean basal serum corticosterone concentrations (A). Main effect of AI or Sex indicated by * (p<0.05), and ** (p<0.01) for circulating IL-4 (B) and IL-5 levels (C). There was a main effect of LB on brainstem serotonin transporter (SERT) (D), hippocampal serotonin receptor 1a (5Htr1a) (E), and hippocampal corticotropin releasing hormone receptor 1 (Crhr1) (F), indicated by * (p<0.05) on E and F, and excluded on D in order to show sex-specific trends of LB and AI/LB on SERT expression, indicted with # (p<0.10). [Contrasts & pairwise group comparisons: Compared to CON mice, AI and AI+LB mice had significantly greater circulating IL-4, AI had greater IL-5 levels, and LB had significantly greater 5Htr1a and Crhr1 expression (p<0.05 and 0.01). Pairwise comparisons revealed no other group differences.]