Novel assays to measure forms of interleukin-33 in the circulation and airway mucosa

Introduction: Interleukin (IL)-33 is released from the airway epithelium and can sense the proteolytic and oxidative environment (Cohen, E.S. et al. Nat Comms 2015; 6:8327; Scott, I.C. et al. Sci Rep 2018; 8:3363). IL-33 amplifies inflammation via its receptor ST2 and is associated with type 2 immunity. IL-33 exists in reduced and oxidized forms of IL-33 but can be sequestered by soluble ST2 (sST2), however, there are no assays that distinguish forms. Inconsistencies with IL-33 detection with commercial assays might be due to the differential detection of these antigenically distinct forms. Here we describe new assays for IL-33 forms and measurements in patient samples. Methods: anti-IL-33 mAb were paired in S-plex assays (MSD) with lower limit of detection (LLOD): reduced IL-33 (~16 fg/ml), oxidized (ox) (~100 fg/ml) or IL-33/sST2 complexes (~100 fg/ml). Assays were evaluated in serum from healthy (n=20), asthmatics (n=14) and COPD patients (n=16) or nasal mucosal lining fluid (MLF) from atopics (n=6) following allergen challenge (NAC, 0-24h). Results: We detected comparable levels of IL-33/sST2 complex (~1-5 pg/ml) in serum from healthy individuals and patients. Total sST2 in serum was similar between patients but at high levels (~10-100 ng/ml). Reduced and oxIL-33 were undetected in serum. In contrast, we detected reduced and oxIL-33 in MLF from atopics with levels increased post-NAC that peaked after 1 h (20-1000 pg/ml). In comparison, PGD2 (1-3 ng/ml) peaked after 15 min and IL-5 (~25-300 pg/ml) after 3-10 h. IL-33/sST2 complexes were low in MLF. Conclusions: We have generated novel assays for detection of IL-33 forms that will provide insights into mechanisms regulating IL-33 activity.