Sex-dependent regulation of mucin gene transcription and airway secretion and mechanics following intra-airway IL-13 in mice with conditional loss of club cell Creb1.

Introduction: Interleukin 13 (IL-13) is an important effector molecule in allergic asthma. IL-13-mediated mucin hypersecretion requires conversion of secretoglobin-positive club cells into goblet cells through suppression of forkhead box A2 (FOXA2) and induction of SAM pointed domain containing ETS transcription factor (SPDEF). IL-13-mediated mucin hypersecretion may also include modulation of purinergic and muscarinic receptors that control basal and stimulated mucin secretion. We recently found that the transcription factor cAMP response element-binding protein (Creb1) inhibits FOXA2 and modulates mucus secretion in mice. Methods: We tested the hypothesis that loss of club cell Creb1 mitigates the pro-mucin effects of IL-13. We challenged male and female mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-13 or vehicle. We also studied human "club cell-like" NCI-H322 cells. Results: Loss of club cell Creb1 augmented IL-13-mediated increases in mRNA for the gel-forming mucins Muc5ac and Muc5b and prevented IL-13-mediated decreases in muscarinic 3 receptor (M3R) mRNA in male airways. In female airways, loss of club cell Creb1 reduced M3R mRNA and significantly blunted IL-13-mediated increases in purinergic receptor P2Y2 (P2ry2) mRNA but did not impact Muc5ac and Muc5b mRNA. Despite changes in mucins and secretion machinery, goblet cell density following cholinergic stimulation was not impacted by loss of club cell Creb1 in either sex. IL-13 treatment decreased basal airway resistance across sexes in mice with loss of club cell Creb1, whereas loss of club cell Creb1 augmented IL-13-mediated increases in airway elastance in response to methacholine. NCI-H322 cells displayed IL-13 signaling components, including IL-13Rα1 and IL-4Rα. Pharmacologic inhibition of CREB reduced IL-13Rα1 mRNA, whereas recombinant CREB decreased IL-4Rα mRNA. Application of IL-13 to NCI-H322 cells increased concentrations of cAMP in a delayed manner, thus linking IL-13 signaling to CREB signaling. Conclusion: These data highlight sex-specific regulation of club cell Creb1 on IL-13-mediated mucin hypersecretion and airway mechanics.

Progressive tauopathy disrupts breathing stability and chemoreflexes during presumptive sleep in mice.

Rationale: Although sleep apnea occurs in over 50% of individuals with Alzheimer's Disease (AD) or related tauopathies, little is known concerning the potential role of tauopathy in the pathogenesis of sleep apnea. Here, we tested the hypotheses that, during presumptive sleep, a murine model of tauopathy (rTg4510) exhibits: 1) increased breathing instability; 2) impaired chemoreflex function; and 3) exacerbation of these effects with tauopathy progression. Methods: rTg4510 mice initially develop robust tauopathy in the hippocampus and cortex, and eventually progresses to the brainstem. Type I and II post-sigh apnea, Type III (spontaneous) apnea, sigh, and hypopnea incidence were measured in young adult (5-6 months; n = 10-14/group) and aged (13-15 months; n = 22-24/group) non-transgenic (nTg), monogenic control tetracycline transactivator, and bigenic rTg4510 mice using whole-body plethysmography during presumptive sleep (i.e., eyes closed, curled/laying posture, stable breathing for >200 breaths) while breathing room air (21% O2). Peripheral and central chemoreceptor sensitivity were assessed with transient exposures (5 min) to hyperoxia (100% O2) or hypercapnia (3% and 5% CO2 in 21% O2), respectively. Results: We report significant increases in Type I, II, and III apneas (all p < 0.001), sighs (p = 0.002) and hypopneas (p < 0.001) in aged rTg4510 mice, but only Type III apneas in young adult rTg4510 mice (p < 0.001) versus age-matched nTg controls. Aged rTg4510 mice exhibited profound chemoreflex impairment versus age matched nTg and tTA mice. In rTg4510 mice, breathing frequency, tidal volume and minute ventilation were not affected by hyperoxic or hypercapnic challenges, in striking contrast to controls. Histological examination revealed hyperphosphorylated tau in brainstem regions involved in the control of breathing (e.g., pons, medullary respiratory column, retrotrapezoid nucleus) in aged rTg4510 mice. Neither breathing instability nor hyperphosphorylated tau in brainstem tissues were observed in young adult rTg4510 mice. Conclusion: Older rTg4510 mice exhibit profound impairment in the neural control of breathing, with greater breathing instability and near absence of oxygen and carbon-dioxide chemoreflexes. Breathing impairments paralleled tauopathy progression into brainstem regions that control breathing. These findings are consistent with the idea that tauopathy per se undermines chemoreflexes and promotes breathing instability during sleep.

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B.

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1ß (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown. Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human "club cell-like" H322 cells. Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling. Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2.