Comparison of Protease Activated Receptor‐2 (PAR‐2) Activity of Eosinophils Derived from Asthmatic and Non‐Asthmatic Puerto Ricans
Authors: Rosado Angely Ortiz , Rivera Marinel Ocasio , Marin‐Maldonado Frances , Trossi‐Torres Geraline , Suarez Edu B 
Published: June 2021

Asthma affects over 18 million adults and 6 million children with an increase of 15% during the last decade. This is a multifactorial, chronic, respiratory, and inflammatory disease with significant social and economic impact. Puerto Ricans either living on the Island or the mainland United States, show the highest prevalence (12–19%), supporting the genetic component in this population. An asthmatic episode is triggered by environmental stimuli: spores, cockroaches, and dust mites allergens, usually containing proteases such as tryptase. Based on the important role of proteases as asthma‐causing allergens, we studied the Protease Activated Receptor (PAR‐2), a G‐protein coupled receptor with the gene locus at 5q 13.3. Interestingly, PAR‐2 location coincides with previously linked candidate region for asthma. PAR‐2 is expressed in granulocytes, human respiratory airway epithelial cells, bronchial vessels, and airway smooth muscle cells, all of which intervene in asthma pathology. The aim of this study was to use human‐derived eosinophils to determine the intracellular calcium levels as indicator of PAR‐2 activation, once exposed to the agonist AC264613. We hypothesized that asthmatic participants will show higher levels of intracellular calcium levels when compared to non‐asthmatic. We conducted a case‐control study from 61 participants (33 asthmatic and 28 non‐asthma; IRB #171205‐ES). The inclusion criteria was: ages 5–62, be at least a third generation Puerto Rican, and be current resident of Puerto Rico. Exclusion criteria included, among other: chronic bronchitis, pulmonary emphysema, and congestive heart failure. We isolated eosinophils from whole blood samples collected in EDTA containing tubes. Once isolated by negative selection, we seeded the eosinophils in a 96‐well plate and cultured overnight under standard conditions. The next day, we treated the eosinophils with the agonist (AC264613:10uM) or the antagonist (FSLLRY‐NH2: 2.5uM), followed by the Fluo‐4 direct calcium assay to determine the eosinophils intracellular calcium levels at 45 seconds after treatment. The data was normalized with the untreated control and analyzed using IBM SPSS v25 and GraphPad Prism 6.01. Our results showed that the studied population was represented by 64.2% female and 35.8 % males with a mean of 30.0 +/− 1.74 years old. There was a significant difference (p< 0.05) of intracellular calcium levels in asthmatic‐derived eosinophils response to agonist when compared to non‐asthmatics (~ 300% increase). This increase of intracellular calcium levels indirectly represents the activation of PAR‐2. We are now evaluating our results by using an eosinophil degranulation assay. To our knowledge, this study represents the first using eosinophils in a case‐control study in asthma and PAR‐2. The obtained outcomes show the potential of PAR‐2 antagonist as adjuvant therapy as a standard of care for this prevalent disease.Support or Funding InformationPuerto Rico Science, Technology and Research Trust to ESM; NIH‐NIGMS #R25GM096955 to ESM; PRCTRC NIMDH and NIAID #U54MD007587 to PHSUThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.