Transgenic mice overexpressing the PGE<sub>2</sub> receptor EP<sub>2</sub> on mast cells exhibit a protective phenotype in a model of allergic asthma
Authors: Adrián Urbano, Judith Plaza, Sandra Turon, Anna Pujol, Cristina Costa‐Farré, Alberto Marco, César Picado, Rosa Torres, Fernando de Mora
Journal: Allergy (2021)

Pharmacological inhibition of airway mast cell (MC) activity has received considerable attention as a plausible anti-asthma strategy. Activation of the MC prostaglandin E2 (PGE2) receptor E prostanoid 2 (EP2) may drive this potentially therapeutic mechanism. Indeed, specific EP2 activation inhibits both murine1 and human2 airway MC activity in vitro. Furthermore, EP2 agonism-mediated reduction in airway pathology has been linked to restricted MC activity in human3 and in murine4 models, and we reported upregulation of PGE2 and of EP2 mRNA, but not EP1, EP3, and EP4, upon exposure of mice to house dust mite (HDM) aeroallergens.5 In light of these data, we hypothesized that the EP2-mediated improvement observed in mouse models of asthma was a consequence of reduced MC activity, and that the PGE2-EP2-MC axis acts as an endogenous protective mechanism.6 To bridge this knowledge gap, we have created transgenic (TG) mice overexpressing the EP2 receptor on MCs and have studied the response of mice sensitized to HDM aeroallergens (protocol in Supp. Figure 1). To generate the TG colony, a construct containing the MC-specific Cma1 promoter and the EP2 receptor coding region (Ptger2) was microinjected into C57BL/6OlaHsd zygotes. The TG mice were backcrossed to obtain mice overexpressing EP2 on a BALB/c genetic background (see details in the Repository Material and Supp. Figures 2-4). All animals were bred at the Animal Facility of the Universidad Autónoma de Barcelona, and the procedures were approved by the Ethics Committee for Animal Research. To our knowledge, this is the first report of a mouse line constitutively overexpressing EP2 selectively on MCs. To assess the impact of MC-specific EP2 overexpression on airway MC activity, murine chemokine mouse MC protease 1 (mMCP1) levels were measured in lung homogenates of mice sensitized to HDM aeroallergens. Non-sensitized wild-type (WT) and TG animals both had virtually no MC activity (Figure 1a), which reflected the equivalent baseline airway MC numbers (Supp. Figure 4a). mMCP1 production increased in HDM-exposed mice of both genotypes. However, mMCP1 upregulation was significantly lower in sensitized TG than in sensitized WT animals (Figure 1a). Therefore, EP2 overexpression reduced HDM-induced MC activation in vivo. To confirm that this effect was specifically driven by EP2 activation, mice were pretreated with the selective EP2 antagonist PF-04418948 prior to sensitization. In both TG and WT animals, EP2 antagonism fostered a further increase of HDM-induced MC activity, along the lines of our recent observation in murine lung MCs in vitro.1 Interestingly, the difference in HDM-induced MC activity observed between TG and WT mice was abrogated after blockade of the EP2 receptor. All in all, this suggests that EP2 receptor overexpression on MCs leads to an increased interation between endogenous PGE2 and EP2, which restricts HDM-induced MC activity. To analyze the consequences of EP2-driven MC inhibition, HDM-induced airway inflammation and reactivity were assessed. Inflammation was quantitated through a differential inflammatory cell count in bronchoalveolar lavage suspensions (Figure 1b). Prominent bronchovascular eosinophilic infiltration developed in mice exposed to HDM. Mimicking the MC activity pattern, the eosinophilia was lower in TG mice than in WT animals (although the difference did not achieve statistical significance). The lower eosinophilic infiltration was paralleled by a trend toward a higher number of macrophages, whereas neutrophil and lymphocyte counts remained equivalent between TG and WT mice (Supp. Figure 5). Also paralleling the effect observed on MCs, both TG and WT animals exhibited a further dose-dependent increase of inflammatory infiltrate in mice pretreated with EP2 antagonist. However, EP2 antagonism did not fully reverse the improvement of the inflammatory process driven by EP2 overexpression. Analyzed in the context of previous data where reduced bronchovascular inflammation was demonstrated in HDM-sensitized mice treated with a selective EP2 agonist,4 the current data support the view that an endogenous PGE2-EP2-MC compensatory mechanism may attenuate HDM-driven eosinophilic inflammation. Finally, invasive plethysmography was used to measure airway reactivity to methacholine (Figure 2). Non-sensitized TG and WT animals exhibited no difference in baseline airway constriction upon increasing doses of methacholine (Figure 2a). HDM-sensitized WT animals developed the expected level of airway hyperresponsiveness (AHR) after a 10-day exposure to HDM aeroallergens. Indeed, as per ANOVA, lung resistance (RL) was significantly higher in sensitized than in non-sensitized WT mice. By contrast, the methacholine dose-response curve of sensitized TG animals overexpressing EP2 on MCs showed no induction of AHR upon exposure to HDM. Indeed, contrary to WT animals, the RL of sensitized and non-sensitized TG mice did not differ. This finding agrees with our previous observations after exogenous EP2 activation.4 Pretreatment of mice with the EP2 antagonist PF-0418948 (Figure 2b) abrogated the beneficial effect exerted by EP2 overexpression on HDM-induced airway reactivity. Contrary to mice that were not pretreated with PF-0418948, both TG and WT animals exhibited similar levels of AHR after methacholine treatment in the presence of 2.5 mg/kg of the antagonist. We have shown that MC-specific EP2 overexpression protects from HDM aeroallergen-induced airway pathology in a IgE-dependent murine asthma model. A direct connection between reduced MC activity and improvement of airway inflammation and reactivity has been demonstrated. The finding that EP2 antagonism worsens HDM-induced harmful effects in WT animals, and reverses the benefit exerted by EP2 overexpression, supports the hypothesis that the PGE2–EP2–MC axis acts as a natural defensive barrier. This is consistent with the recent observation that PGE2 protects from the development of anaphylaxis and MC hyperresponsiveness.7 It also builds on our hypothesis of compensatory PGE2 and EP2 upregulation in mice exposed to HDM5 and on the preventive effect exerted by exogenous EP2 activation.4 In a recent consensus report,8 the European Academy of Allergy and Clinical Immunology (EAACI) Task Force discussed the need to further research the role of eicosanoids in asthma and allergic diseases. The authors specifically mention the bronchoprotective and anti-inflammatory properties of PGE2 in the lungs, remind us of its ability to reduce MC activity, and request further studies with selective EP1-4 analogues. By using an alternative approach, we hope to have contributed to the link between eicosanoid biology and asthma pathophysiology. Further characterization of the cellular and molecular events involved in the PGE2–EP2–MC axis in the newly created C.B6-Tg (Cma1-Ptger2) mice will undoubtedly unravel new anti-asthma therapeutic target candidates and offer future research opportunities. This study was supported by a grant from Instituto de Salud Carlos III of the Spanish Ministry of Science, Innovation and Universities (Ref. PI18/01702). The authors declare that they have no conflict of interest. Urbano A involved in induction of mice's sensitization and conduction of airway hyperreactivity and inflammation studies, as well as mast cell activity assessment; data analysis and interpretation; and manuscript writing. Plaza J contributed to early stage airway hyperreactivity and inflammation studies in HDM-sensitized mice, as well as mast cell activity assessment. Turon S contributed to transgenic mice colony generation and handling. Pujol A contributed to transgenic mice colony generation and handling; manuscript revision. Costa-Farré C contributed to mice handling for airway hyperreactivity studies. Marco A involved in assessment of airway inflammation and interpretation. Picado C involved in study design and data interpretation, and manuscript revision. Torres R involved in experiment supervision and support in transgenic mice colony generation; data analysis and interpretation; and manuscript revision. de Mora F involved in study design, experiment supervision, and data analysis and interpretation; manuscript writing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.