Direct targeting of STAT3 by xanthatin suppresses allergic airway inflammation via inhibition of TSLP release and NK2 cell polarization

Ethnopharmacological relevance: Xanthatin is a natural sesquiterpene lactone derived from Xanthium strumarium L. (cang-er-zi) in traditional Chinese medicine (TCM), which has been reported with multiple pharmacological activities. Although the bioactivity of xanthatin against asthma is well recognized, the underlying mechanism lacks thorough research.

Aim of the study: To systematically investigate the therapeutic potential and mechanism of xanthatin ameliorating allergic airway inflammation.

Materials and methods: The alleviating capacity of xanthatin was evaluated on a 24-day house dust Mite (HDM)-induced mice asthma model. Airway responsiveness was determined by detecting enhanced pause (penh) upon stimulation with different concentrations of methacholine chloride. IgE in serum, and protein levels of IL-4, IL-5, IL-13, IL-25, IL-33, thymic stromal lymphopoietin (TSLP) and IFN-γ in lungs were detected by enzyme-linked immunosorbent assay (ELISA). Lung histological changes were assessed after tissue slices were stained with haematoxylin and eosin (H&E). Changes of subsets of natural killer cells (NK) in mice peripheral blood and human NK92MI cell line were analyzed by flow cytometry. Anti-Asialo GM1 antibody was applied for the depletion of NK, and the key role of NK2 involved in asthma was determined by adoptive transfer. To validate the pivotal role of TSLP in inducing NK2 polarization, anti-TSLP monoclonal antibody was used. The signal transducer and activator of transcription 3 (STAT3) inhibitor stattic, alongside with flow cytometry and western blotting, was applied to analyze whether STAT3 activation was the indispensable part for TSLP to induce NK2 and bronchial epithelial cell to produce TSLP. Molecular docking, microscale thermophoresis (MST), and surface plasmon resonance (SPR) assays were applied to test the direct binding of xanthatin to STAT3.

Results: Xanthatin administration significantly alleviated hallmark asthmatic features, including airway hyperresponsiveness, eosinophilia, elevated serum IgE and high levels of Th2 cytokines in airway, and lung tissue pathological changes. Flow cytometry analysis revealed that xanthatin specifically reduced the lung accumulation of IL-13-producing NK2, but not IFN-γ⁺ NK1. Depletion of NK cells and adoptive transfer of NK2 cells confirmed the pivotal role of NK2 cells in driving the pathology and in mediating xanthatin's protective effects. Mechanistically, xanthatin selectively suppressed bronchial epithelial cell-derived TSLP both in vivo and in vitro. We identified TSLP as a novel inducer of STAT3-dependent NK2 cell differentiation, which was inhibited by xanthatin. Furthermore, xanthatin directly bound to STAT3 protein with high affinity. Consequently, xanthatin inhibited STAT3 phosphorylation in both bronchial epithelial cells (suppressing TSLP production) and in NK cells (impairing TSLP-induced NK2 differentiation).

Conclusions: Xanthatin ameliorates allergic airway inflammation by directly targeting STAT3, thereby dually inhibiting the TSLP/STAT3 axis in epithelial cells and the subsequent STAT3-dependent differentiation of pathogenic NK2 cells, presenting a promising multi-targeted strategy for asthma treatment.