3196 | LETTERS 5 drugs in the investigation of cutaneous adverse drug reactions. Department of Infectious Disease, Bordeaux University Contact Dermatitis. 2001;45(6):321-­328. Hospitals, Bordeaux, France 2. Rosenblatt AE, Stein SL. Cutaneous reactions to vaccinations. Clin 6 Dermatol. 2015;33(3):327-­332. Department of Anatomopathology, Bordeaux University 3. Corbeddu M, Diociaiuti A, Vinci MR, et al. Transient cutaneous Hospitals, Bordeaux, France 7 manifestations after administration of Pfizer-­BioNTech COVID-­19 Research Unit INSERM U1035, Bordeaux, France Vaccine: an Italian single centre case series. J Eur Acad Dermatol Venereol. 2021. https://doi.org/10.1111/jdv.17268 Correspondence 4. Banerji A, Wickner PG, Saff R, et al. mRNA Vaccines to Prevent Anne-­Sophie Darrigade, Dermatology Department, Saint-­ COVID-­ 19 Disease and Reported Allergic Reactions: Current Evidence and Suggested Approach. J Allergy Clin Immunol Pract. André Hospital, 1, rue Jean Burguet 33000 Bordeaux, 2020;S2213–­2198(20):31411-­31412. France. 5. Bianchi L, Biondi F, Hansel K, Murgia N, Tramontana M, Stingeni L. Email: anne-sophie.darrigade@chu-bordeaux.fr Skin tests in urticaria/angioedema and flushing to Pfizer-­BioNTech SARS-­CoV-­2 vaccine: Limits of intradermal testing. Allergy. 2021;76: 2605-2607. https://doi.org/10.1111/all.14839 ORCID 6. Pedrosa AF, Morais P, Nogueira A, Pardal J, Azevedo F. Sweet's syn- Anne-­Sophie Darrigade https://orcid.org/0000-0003-4034-0395 drome triggered by pneumococcal vaccination. Cutan Ocul Toxicol. 2013;32(3):260-­261. REFERENCES 7. Ratzinger G, Burgdorf W, Zelger BG, Zelger B. Acute febrile neutro- philic dermatosis: a histopathologic study of 31 cases with review 1. Barbaud A, Gonçalo M, Bruynzeel D, Bircher A. European Society of literature. Am J Dermatopathol. 2007;29(2):125-­133. of Contact Dermatitis. Guidelines for performing skin tests with DOI: 10.1111/all.14988 Transgenic mice overexpressing the PGE2 receptor EP2 on mast cells exhibit a protective phenotype in a model of allergic asthma To the Editor, region (Ptger2) was microinjected into C57BL/6OlaHsd zygotes. The Pharmacological inhibition of airway mast cell (MC) activity has re- TG mice were backcrossed to obtain mice overexpressing EP2 on a ceived considerable attention as a plausible anti-­asthma strategy. BALB/c genetic background (see details in the Repository Material Activation of the MC prostaglandin E2 (PGE2) receptor E prostanoid and Supp. Figures 2-­4). All animals were bred at the Animal Facility 2 (EP2) may drive this potentially therapeutic mechanism. Indeed, of the Universidad Autónoma de Barcelona, and the procedures specific EP2 activation inhibits both murine1 and human2 airway were approved by the Ethics Committee for Animal Research. To our MC activity in vitro. Furthermore, EP2 agonism-­ mediated reduc- knowledge, this is the first report of a mouse line constitutively over- tion in airway pathology has been linked to restricted MC activity expressing EP2 selectively on MCs. in human3 and in murine 4 models, and we reported upregulation of To assess the impact of MC-­specific EP2 overexpression on air- PGE2 and of EP2 mRNA, but not EP1, EP3, and EP4, upon exposure of way MC activity, murine chemokine mouse MC protease 1 (mMCP1) mice to house dust mite (HDM) aeroallergens.5 In light of these data, levels were measured in lung homogenates of mice sensitized to we hypothesized that the EP2-­mediated improvement observed in HDM aeroallergens. Non-­sensitized wild-­t ype (WT) and TG animals mouse models of asthma was a consequence of reduced MC activity, both had virtually no MC activity (Figure 1A), which reflected the and that the PGE2-­EP2-­MC axis acts as an endogenous protective equivalent baseline airway MC numbers (Supp. Figure 4A). mMCP1 6 mechanism. To bridge this knowledge gap, we have created trans- production increased in HDM-­ exposed mice of both genotypes. genic (TG) mice overexpressing the EP2 receptor on MCs and have However, mMCP1 upregulation was significantly lower in sensitized studied the response of mice sensitized to HDM aeroallergens (pro- TG than in sensitized WT animals (Figure 1A). Therefore, EP2 over- tocol in Supp. Figure 1). To generate the TG colony, a construct con- expression reduced HDM-­induced MC activation in vivo. To confirm taining the MC-­specific Cma1 promoter and the EP2 receptor coding that this effect was specifically driven by EP2 activation, mice were This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n-­NonCo​mmerc​ial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd. LETTERS | 3197 F I G U R E 1 (A) HDM-­induced airway MC activity in mice overexpressing EP2. Mast cell (MC) activity was assessed by measuring mMCP1 concentration in lung homogenates normalized by total protein. WT mice exposed to HDM exhibit a significant increase in MC activity. The level of MC activation increased further in the presence of an E prostanoid receptor 2 (EP2) antagonist (PF) at both 2.5 and 10 mg/kg. TG mice overexpressing EP2 on MC exhibited a less pronounced increase of MC activity. Pretreatment with an EP2 antagonist counteracted the reduced MC activity in TG mice, resulting in activity levels that were essentially equivalent between both genotypes (TG and WT). (B) HDM-­induced eosinophilia in mice overexpressing EP2. Airway inflammation was assessed by a differential cell count in bronchoalveolar lavage suspensions (see the full inflammatory cell count in Supp. Figure 5). WT mice exposed to HDM exhibited significant eosinophilia, which further increased in the presence of an EP2 antagonist at both 2.5 and 10 mg/kg. In TG mice overexpressing EP2 on MC, eosinophil recruitment was attenuated. After pretreatment with the EP2 antagonist, the effect of EP2 overexpression on eosinophilia was abrogated. Results are expressed as mean ± SEM. Statistical significance was evaluated using a two-­way analysis of variance (ANOVA) with a Bonferroni post hoc test. *p<0.05, **p<0.01, and ***p<0.001. HDM (house dust mite-­sensitized); MC (mast cell); NS (non-­sensitized); PF (PF-­0 441848); TG (transgenic); Veh (vehicle); and WT (wild-­t ype) F I G U R E 2 HDM-­induced airway reactivity in mice overexpressing EP2. Airway reactivity was assessed by means of invasive plethysmography and expressed as a percentage of baseline lung resistance (RL). (A) Airway resistance in WT and TG mice exposed to HDM. Sensitization to HDM aeroallergens induced a significant increase in airway reactivity to methacholine (ie, airway hyperresponsiveness [AHR]) in WT mice (HDM/WT), while sensitized TG mice overexpressing E prostanoid receptor 2 (EP2) (HDM/TG) did not develop AHR. (B) Airway reactivity after pretreatment with an EP2 antagonist. In the presence of the EP2 antagonist (PF), both sensitized WT (HDM/WT+PF) and sensitized transgenic (HDM/TG+PF) mice developed AHR. Indeed, contrary to what is observed in Figure 2A, there was no significant difference between HDM/WT and HDM/TG when the mice were pretreated with 2.5 mg/kg of PF. Results are expressed as mean ± SEM. Statistical significance was evaluated using a two-­way analysis of variance (ANOVA) with a Bonferroni post hoc test. Statistically significant differences were found between baseline reactivity (NS mice) and HDM-­sensitized mice. HDM (house dust mite-­sensitized); RL (lung resistance); NS (non-­sensitized); PF (PF-­0 441848); TG (transgenic); and WT (wild-­t ype) 3198 | LETTERS pretreated with the selective EP2 antagonist PF-­0 4418948 prior to and reverses the benefit exerted by EP 2 overexpression, supports sensitization. In both TG and WT animals, EP2 antagonism fostered a the hypothesis that the PGE 2–­EP 2–­MC axis acts as a natural de- further increase of HDM-­induced MC activity, along the lines of our fensive barrier. This is consistent with the recent observation that recent observation in murine lung MCs in vitro.1 Interestingly, the PGE 2 protects from the development of anaphylaxis and MC hy- difference in HDM-­induced MC activity observed between TG and perresponsiveness.7 It also builds on our hypothesis of compen- WT mice was abrogated after blockade of the EP2 receptor. All in satory PGE 2 and EP 2 upregulation in mice exposed to HDM5 and all, this suggests that EP2 receptor overexpression on MCs leads to on the preventive effect exerted by exogenous EP 2 activation.4 an increased interation between endogenous PGE2 and EP2, which In a recent consensus report, 8 the European Academy of Allergy restricts HDM-­induced MC activity. and Clinical Immunology (EAACI) Task Force discussed the need To analyze the consequences of EP2-­ driven MC inhibition, to further research the role of eicosanoids in asthma and allergic HDM-­induced airway inflammation and reactivity were assessed. diseases. The authors specifically mention the bronchoprotective Inflammation was quantitated through a differential inflamma- and anti-­inflammatory properties of PGE 2 in the lungs, remind us tory cell count in bronchoalveolar lavage suspensions (Figure 1B). of its ability to reduce MC activity, and request further studies Prominent bronchovascular eosinophilic infiltration developed in with selective EP1-­4 analogues. By using an alternative approach, mice exposed to HDM. Mimicking the MC activity pattern, the eo- we hope to have contributed to the link between eicosanoid biol- sinophilia was lower in TG mice than in WT animals (although the ogy and asthma pathophysiology. Further characterization of the difference did not achieve statistical significance). The lower eosino- cellular and molecular events involved in the PGE2–­EP 2–­MC axis philic infiltration was paralleled by a trend toward a higher number of in the newly created C.B6-­Tg (Cma1-­P tger2) mice will undoubtedly macrophages, whereas neutrophil and lymphocyte counts remained unravel new anti-­asthma therapeutic target candidates and offer equivalent between TG and WT mice (Supp. Figure 5). Also parallel- future research opportunities. ing the effect observed on MCs, both TG and WT animals exhibited a further dose-­dependent increase of inflammatory infiltrate in mice F U N D I N G I N FO R M AT I O N pretreated with EP2 antagonist. However, EP2 antagonism did not Instituto de Salud Carlos III, Grant/Award Number: PI18/01702 fully reverse the improvement of the inflammatory process driven by EP2 overexpression. Analyzed in the context of previous data where AC K N OW L E D G M E N T S reduced bronchovascular inflammation was demonstrated in HDM-­ This study was supported by a grant from Instituto de Salud Carlos sensitized mice treated with a selective EP2 agonist,4 the current III of the Spanish Ministry of Science, Innovation and Universities data support the view that an endogenous PGE2-­EP2-­MC compensa- (Ref. PI18/01702). tory mechanism may attenuate HDM-­driven eosinophilic inflamma- tion. Finally, invasive plethysmography was used to measure airway C O N FL I C T O F I N T E R E S T reactivity to methacholine (Figure 2). Non-­sensitized TG and WT The authors declare that they have no conflict of interest. animals exhibited no difference in baseline airway constriction upon increasing doses of methacholine (Figure 2A). HDM-­sensitized WT AU T H O R C O N T R I B U T I O N S animals developed the expected level of airway hyperresponsive- Urbano A involved in induction of mice's sensitization and conduc- ness (AHR) after a 10-­day exposure to HDM aeroallergens. Indeed, tion of airway hyperreactivity and inflammation studies, as well as as per ANOVA, lung resistance (RL) was significantly higher in sensi- mast cell activity assessment; data analysis and interpretation; and tized than in non-­sensitized WT mice. By contrast, the methacholine manuscript writing. Plaza J contributed to early stage airway hyper- dose-­response curve of sensitized TG animals overexpressing EP2 on reactivity and inflammation studies in HDM-­sensitized mice, as well MCs showed no induction of AHR upon exposure to HDM. Indeed, as mast cell activity assessment. Turon S contributed to transgenic contrary to WT animals, the RL of sensitized and non-­sensitized TG mice colony generation and handling. Pujol A contributed to trans- mice did not differ. This finding agrees with our previous observa- genic mice colony generation and handling; manuscript revision. 4 tions after exogenous EP2 activation. Pretreatment of mice with Costa-­Farré C contributed to mice handling for airway hyperreac- the EP2 antagonist PF-­0 418948 (Figure 2B) abrogated the beneficial tivity studies. Marco A involved in assessment of airway inflamma- effect exerted by EP2 overexpression on HDM-­induced airway reac- tion and interpretation. Picado C involved in study design and data tivity. Contrary to mice that were not pretreated with PF-­0 418948, interpretation, and manuscript revision. Torres R involved in experi- both TG and WT animals exhibited similar levels of AHR after metha- ment supervision and support in transgenic mice colony generation; choline treatment in the presence of 2.5 mg/kg of the antagonist. data analysis and interpretation; and manuscript revision. de Mora F We have shown that MC-­ specific EP 2 overexpression pro- involved in study design, experiment supervision, and data analysis tects from HDM aeroallergen-­induced airway pathology in a IgE-­ and interpretation; manuscript writing. dependent murine asthma model. A direct connection between reduced MC activity and improvement of airway inflammation Adrián Urbano1 and reactivity has been demonstrated. The finding that EP2 an- Judith Plaza1 tagonism worsens HDM-­induced harmful effects in WT animals, Sandra Turon2 LETTERS | 3199 Anna Pujol2 César Picado https://orcid.org/0000-0001-7400-4993 3 Cristina Costa-­Farré Rosa Torres https://orcid.org/0000-0002-9109-4035 Alberto Marco3 Fernando de Mora https://orcid.org/0000-0002-3002-6004 César Picado 4,5 Rosa Torres1 REFERENCES 1 Fernando de Mora 1. Plaza J, Torres R, Urbano A, Picado C, de Mora F. In vitro and in vivo validation of EP2-­receptor agonism to selectively achieve inhibi- 1 tion of mast cell activity. Allergy Asthma Immunol Res. 2020;12:712. Department of Pharmacology, Therapeutics and Toxicology, https://doi.org/10.4168/aair.2020.12.4.712 Universitat Autònoma de Barcelona, Barcelona, Spain 2. Kay LJ, Yeo WW, Peachell PT. Prostaglandin E 2 activates EP 2 re- 2 Transgenic Animal Unit, Center of Animal Biotechnology and ceptors to inhibit human lung mast cell degranulation. Br J Pharmacol. Gene Therapy, Universitat Autònoma de Barcelona, Bellaterra 2006;147:707-­713. https://doi.org/10.1038/sj.bjp.0706664 3. Säfholm J, Manson ML, Bood J, et al. Prostaglandin E 2 inhibits (Barcelona), Spain 3 mast cell –­dependent bronchoconstriction in human small airways Department of Surgery and Animals Medicine, Universitat through the E prostanoid subtype 2 receptor. J Allergy Clin Immunol. Autònoma de Barcelona, Barcelona, Spain 2015;136:1232-­1240. https://doi.org/10.1016/j.jaci.2015.04.002 4 4. Serra-­Pages M, Torres R, Plaza J, et al. Activation of the Prostaglandin Department of Pneumology and Respiratory Allergy, Hospital E2 receptor EP2 prevents house dust mite-­induced airway hyperres- Clínic, IDIBAPS (Institut d’Investigacions Biomèdiques August Pi ponsiveness and inflammation by restraining mast cells’ activity. Clin i Sunyer), Universitat de Barcelona, Barcelona, Spain Exp Allergy. 2015;45:1590-­1600. https://doi.org/10.1111/cea.12542 5 CIBERES (Centro de Investigación Biomédica en Red de 5. Herrerias A, Torres R, Serra M, et al. Activity of the cyclooxy- Enfermedades Respiratorias), Barcelona, Spain genase 2-­ prostaglandin-­ E prostanoid receptor pathway in mice exposed to house dust mite aeroallergens, and impact of ex- ogenous prostaglandin E2. J Inflamm. 2009;6:1-­ 9. https://doi. Correspondence org/10.1186/1476-­9255-­6-­3 0 Fernando de Mora, Department of Pharmacology, 6. Torres R, Picado C, de Mora F. The PGE2-­EP2-­mast cell axis: an Therapeutics and Toxicology, Universitat Autònoma de antiasthma mechanism. Mol Immunol. 2015;63:61-­68. https://doi. Barcelona, Barcelona, Spain. org/10.1016/j.molimm.2014.03.007 7. Rastogi S, Willmes DM, Nassiri M, Babina M, Worm M. PGE2 Email: fernando.demora@uab.cat deficiency predisposes to anaphylaxis by causing mast cell hyper-­responsiveness. J Allergy Clin Immunol. 2020. https://doi. Rosa Torres and Fernando de Mora have contributed equally to this org/10.1016/j.jaci.2020.03.046 study. 8. Sokolowska M, Enrico RG, Diamant Z, et al. Current perspective on eicosanoids in asthma and allergic diseases: EAACI Task Force con- sensus report, part I. Allergy Eur J Allergy Clin Immunol. 2021;76:114-­ ORCID 130. https://doi.org/10.1111/all.14295 Adrián Urbano https://orcid.org/0000-0002-4107-1644 Judith Plaza https://orcid.org/0000-0003-2217-3095 Sandra Turon https://orcid.org/0000-0001-9526-8584 S U P P O R T I N G I N FO R M AT I O N Anna Pujol https://orcid.org/0000-0002-3484-598X Additional supporting information may be found online in the Cristina Costa-­Farré https://orcid.org/0000-0002-0224-6747 Supporting Information section. Alberto Marco https://orcid.org/0000-0003-1680-0822 DOI: 10.1111/all.14997 Type 2 biomarker expression (FeNO and blood eosinophils) is higher in severe adult-­onset than in severe early-­onset asthma To the Editor, 2 biomarkers differs between severe early-­onset asthma (SEA) and Severe asthma is predominantly associated with a type 2 inflamma- severe adult-­onset asthma (SAA). This issue is of particular impor- 1 tory pattern ; however, it is unclear whether the expression of type tance for the question whether the age of asthma onset can be a Abbreviations: FeNO, fractional exhaled nitric oxide; GINA, global initiative for asthma; IgE, immunoglobulin E; OCS, oral corticosteroid; SAA, severe adult-­onset asthma; SEA, severe early-­onset asthma; UK, United Kingdom; UKSAR, United Kingdom severe asthma registry.