Rationale: Airway autoantibodies induce histone-coated eosinophil extracellular traps (EETs). EETs are associated with free eosinophil granules (FEGs) and galectin-10 which autocrystallize ex vivo to form Charcot-Leyden crystals (CLCs).

Objective: To investigate cytokines and autoimmune responses in patients with CLCs.

Methods: Between 2020-2022, CLCs and cell differentials were routinely reported for processed sputum. Cell-free supernatants were assessed for antinuclear antibodies (HUMAN Diagnostics, PMID 28751233), cytokines (Ella^TM, Protein Simple), and danger-associated molecular patterns (DAMPs): high mobility box group 1 (HMGB1) and galectin-10 proteins (Novus Biologicals).

Results: CLCs were reported in 61/3075 (2%) processed sputa slides (protocol: PMID 8804934). All 46 CLC⁺ samples with adequate volume and confirmed severe asthma diagnosis were eosinophilic (?3% eos±FEGs) and comprised the Eos⁺CLC⁺ group. Additionally, Eos⁺CLC^- (n=53) and Eos^-CLC^- (n=32) were included for analysis. Eos⁺CLC⁺ had increased ANAs vs CLC^- groups (P<0.005, Fig 1A). Reactivities against anti-histone IgG were higher in Eos⁺CLC⁺ (96%) vs. Eos^-CLC^- (30%) (Fig 1B). Eos⁺CLC⁺ had higher T2 inflammation (IL-4, IL-5, IL-13, P<0.05), HMGB1 (Fig 1C), and decreased trend for ?free? galactin-10 compared to CLC^- sputa.

Conclusions: We report that severe asthma patients with CLCs have high autoimmune burden marked by DAMPs and autoantibodies.