Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid

Abstract

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

Fig. 1.

Rapid and acute acidification of the airway epithelia in vitro. pH time course of a 1% acetic acid/saline solution or saline solution was applied directly to cultured tracheal airway epithelia derived from male (A) or female (B) piglets. Acid or saline were applied directly after baseline measurements (indicated by dashed line), and pH was recorded within 1 min of application. C: basal pH measurements were not different among groups or sexes before the addition of saline or acid. For all groups, n = 4 cultures derived from four piglets. For A, treatment (F_6,36 = 9.9; P < 0.0001), time (F_1,6 = 26.2; P = 0.002), and interaction (F_6,36 = 7.1; P < 0.0001). For B, treatment (F_6,36 = 15.8; P < 0.0001), time (F_1,6 = 2.0; P = 0.20), and interaction (F_{6, 36} = 12.3; P < 0.0001). For C, treatment (F_1,12 = 0.16; P = 0.69) and sex (F_1,12 = 2.7; P = 0.13). For A and B, *P < 0.05 compared with saline-treated controls. Sal, saline.

Fig. 2.

Intra-airway acid induces key features of asthma in neonatal piglets. A: basal airway resistance, treatment (F_1,20 = 0.16; P = 0.69) and sex (F_1,20 = 0.75; P = 0.89. B: maximal airway resistance in response to increasing doses of intravenous methacholine in male [treatment (F_1,50 = 7.89; P = 0.0071) and dose (F_4,50 = 6.11; P = 0.0004)] and female (C) acid-challenged piglets [treatment (F_1,50 = 1.19; P = 0.28) and dose (F_4,50 = 7.05; P = 0.0001)]. D: mean airway obstruction score is shown; Kruskal-Wallis statistic = 17.22; P = 0.006. E: representative photomicrograph of PAS-stained male saline-challenged piglet and male acid-challenged piglet lung tissue (F) The arrow highlights an airway, whereas the asterisk highlights airway lumen. Scale bar: 1,000 µm. G: muc5AC mRNA in the whole trachea. For males, t = 0.28, df = 10; P = 0.78; for females, t = 0.85, df = 10; P = 0.41. H: muc5AC mRNA in the whole lung. For males, t = 0.80, df = 10; P = 0.44; for females, t = 0.73, df = 10; P = 0.48. I: muc5B mRNA in the whole trachea. For males, t = 2.12, df = 10; P = 0.051; for females, t = 0.81, df = 10; P = 0.44. J: muc5B mRNA in the whole lung. For males, t = 0.86, df = 10; P = 0.41; for females, t = 1.32, df = 10; P = 0.21. K: foxj1 mRNA in the whole trachea. For males, t = 2.31, df = 10; P = 0.043; for females, t = 0.09, df = 10; P = 0.92. L: foxj1 mRNA in the whole lung. For males, t = 1.08, df = 10; P = 0.30; for females, t = 0.55, df = 10; P = 0.59. Data are expressed relative to sex-matched controls, and values are expressed as means ± SE. n = 6 piglets/group. R, resistance; Sal, saline.

Fig. 3.

Inflammation assessment in neonatal piglets challenged with acid. A: number of cells per milliliter in bronchoalveolar lavage fluid is shown. Treatment (F_1,20 = 0.038; P = 0.85) and sex (F_1,20 = 1.04; P = 0.32). B: percentage (%) of cells of granulocytes is given. Treatment (F_1,20 = 1.40; P = 0.25) and sex (F_1,20 = 5.0; P = 0.037). C: bronchoalveolar lavage concentrations of TNF-α. Treatment (F_1,20 = 1.67; P = 0.21) and sex (F_1,20 = 2.52; P = 0.13). D: bronchoalveolar lavage concentrations of IL-13. Treatment (F_{1, 20} = 1.61; P = 0.22) and sex (F_1,20 = 0.37; P = 0.55). E: bronchoalveolar lavage concentrations of IL-17A. Treatment (F_1,20 = 1.29; P = 0.27) and sex (F_1,20 = 0.37; P = 0.13). F: bronchoalveolar lavage concentrations of IL-2 and treatment (F_1,20 = 0.89; P = 0.35) and sex (F_1,20 = 2.54; P = 0.12). G: bronchoalveolar lavage concentrations of IL-10. Treatment (F_1,20 = 1.14; P = 0.29) and sex (F_1,20 = 1.2; P = 0.29). H: bronchoalveolar lavage concentrations of IL-4. Treatment (F_1,20 = 0.032; P = 0.86) and sex (F_1,20 = 4.03; P = 0.0585). I: bronchoalveolar lavage concentrations of IL-1β. Treatment (F_1,20 = 4.61; P = 0.043) and sex (F_1,20 = 0.91; P = 0.35). J: bronchoalveolar lavage concentrations of IL-8. Treatment (F_1,20 = 1.39; P = 0.25) and sex (F_1,20 = 3.58; P = 0.073). K: bronchoalveolar lavage concentrations of IL-6. Treatment (F_1,20 = 3.11; P = 0.0.093) and sex (F_1,20 = 3.79; P = 0.066). n = 6 piglets/group Sal, saline.

Fig. 4.

Sex- and tissue-specific transcriptional responses in acid-challenged piglets. A: illustration demonstrating a simple airway-nervous system circuit. Airway sensory nerves (orange) from the nodose ganglia innervate the airway epithelia and relay information to the brain stem. Upon additional processing of that the information, the parasympathetic nervous system is engaged (blue). Volcano plots demonstrate differentially expressed transcripts using P < 0.05 criteria in acid-challenged piglets relative to sex-matched controls for the male epithelia (B), female epithelia (C), male nodose ganglia (D), female nodose ganglia (E), male brain stem (F), and female brain stem (G). n = 3 piglets each group.

Fig. 5.

Shared and unique transcriptional responses between male and female acid-challenged piglets. Venn diagram demonstrates unique and shared transcripts that increased or decreased in the epithelial transcriptome of male and female piglets (A), in the nodose ganglia transcriptome of male and female piglets (B), and in the brain stem of male and female piglets (C). Venn diagram demonstrates unique and shared biological pathways from overrepresented transcripts that increased or decreased in the epithelia of male and female piglets (D) in the nodose ganglia of male and female piglets (E), and in the brain stem of male and female piglets (G). For all panels, n = 3 piglets each group. M, male; F, female.

Fig. 6.

Nerves in the tracheal airway epithelia in neonatal piglets. Representative photomicrographs of the tracheal epithelia demonstrating nerves (shown in red and highlighted with white arrows) in the tracheal epithelium. The serosal side is shown. n = two female control piglets (A–F) and two male control piglets (G–L). Scale bar: 60 μm and applies to all panels. The epithelial layer is thick and undulates due to the glands attached. This creates extra background and difficulty imaging.

Fig. 7.

Parallel transcriptional responses across tissue compartments in acid-challenged piglets. Heat map demonstrating transcripts that changed in both the airway epithelia and nodose ganglia of male acid-challenged piglets (A) the nodose ganglia and brain stem of male acid-challenged piglets (B), the brain stem and airway epithelia of male acid-challenged piglets (C), the airway epithelia and nodose ganglia of female acid-challenged piglets (D), the nodose ganglia and brain stem of female acid-challenged piglets (E), and the brain stem and airway epithelia of female acid-challenged piglets (F). For all panels, the scale shows log2 fold change. n = 3 piglets each group. For E, there are two stat1 molecules shown because there were two separate ensemble identifiers that changed in parallel, both matching to stat1 (ENSSSCG00000027918 and ENSSSCG00000016057).