Background: The secretion of alarmin cytokines by epithelial cells, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33, initiates inflammatory cascades in asthma. However, alarmin cytokine expression in the upper airways in asthma remains largely unknown. Methods: We recruited 40 participants with asthma into four groups as per the Global Initiative for Asthma (GINA) steps (10 in each group of GINA 1/2, 3, 4, and 5). Cells were derived from nasal, buccal, and throat brushings. Intracellular cytokine expression (TSLP, IL-25, and IL-33) was assessed by flow cytometry in cytokeratin 8⁺ (Ck8⁺) epithelial cells immediately following collection. Results: TSLP was significantly increased (p < 0.001) in GINA 5 patients across nasal, buccal, and throat Ck8⁺ epithelial cells, while IL-25 was elevated in nasal and throat samples (p < 0.003), and IL-33 levels were variable, compared with GINA 1–4 patients. Individual GINA subgroup comparison showed that TSLP levels in nasal samples from GINA 5 patients were significantly (p = 0.03) elevated but did not differ between patients with and without nasal comorbidities. IL-25 and IL-33 (obtained from nasal, buccal, and throat samples) were not significantly different in individual groups. Conclusions: Our study demonstrates for the first time that Ck8⁺ nasal epithelial cells from GINA 5 asthma patients express elevated levels of TSLP.

Background: The secretion of alarmin cytokines by epithelial cells, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33, heralds the onset of the inflammatory cascade and immune effector infiltration in asthma. However, alarmin cytokine expression in the upper airways in asthma remains largely unknown. Methods: We recruited 40 participants with asthma who were categorized into four severity groups as per the Global Initiative for Asthma (GINA) classifications (10 in each group of GINA-1/2, -3, -4, and -5). Cells were derived from buccal, nasal and throat brushings, and intracellular alarmin cytokine expression (TSLP, IL-25, and IL-33) was assessed in cytokeratin 8+ (Ck8+) epithelial cells immediately after collection using flow cytometry with fluorescence minus one (FMO) controls. We assessed differences in alarmin cytokine expressions across asthma severity using quantile regression adjusted for age and sex. Results: Of all patients, 24 (60%) were females with a mean (standard deviation [SD]) age of 41 (16) years. TSLP levels in Ck8+ epithelial cells in nasal samples of GINA-5 patients were significantly (p=0.03) higher than other GINA groups after adjusting for age and sex but did not differ between patients with and without nasal comorbidities. However, we did not find any significant changes in TSLP levels in Ck8+ epithelial cells in buccal and throat samples across GINA groups. IL-25 or IL-33 (obtained from nasal, buccal, and throat epithelial cell samples) were not significantly different across GINA groups. Conclusions: Our study demonstrates for the first time that Ck8+ nasal epithelial cells from GINA-5 asthmatics express elevated levels of TSLP.

Summary: Treatment with benralizumab significantly reduced nasal TSLP in patients with severe asthma. Nasal TSLP shows potential as a pharmacodynamic biomarker, pending validation in larger, randomized studies.

Introduction: The secretion of alarmin cytokines thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33, heralds the onset of the asthma inflammatory cascade. We have shown that TSLP and IL-25 are elevated in nasal epithelia of severe asthmatics. We hypothesize nasal alarmin cytokine expression decreases in response to tezepelumab treatment. Methods: Nasal brushings were collected from candidates for tezepelumab therapy upon diagnosis with severe asthma (n=15). Follow up was done 3 months after first dose. Alarmin cytokines were assessed by flow cytometry using cytokine 8⁺ cells labeled with antibodies against TSLP, IL-25, and IL-33, and analyzed using a Fortessa-SORP flow cytometer. Analysis was performed using multivariable generalized estimating equations with a Gaussian family and an identity link function, along with an exchangeable correlation matrix. Models were adjusted by age, sex, BMI, smoking status, atopy, comorbidities, and chronic rhinosinusitis (CRS). Results: At 3-month follow-up visits, patients exhibited reductions in nasal TSLP, IL-25 and IL-33 expression. Compared to baseline, TSLP expression decreased by 70% (adj. p = 0.004), IL-25 expression decreased by 69% (adj. p < 0.001), and IL-33 expression was reduced by 50% (adj. p = 0.01). In sensitivity analyses, we did not observe collinearity between covariates among comorbidities, atopy, and CRS. Conclusions: Biologic therapy was associated with reduced nasal epithelial expression of TSLP, IL-25 and IL-33, seen as early as two doses after therapy initiation. Our data suggests depletion of TSLP is linked to reduced expression of IL-25 and IL-33. Further work is needed to elucidate this feedback mechanism and understand its implications for severe asthma.