Immune memory has been expanded to group 2 innate lymphoid cells (ILC2s), but the cellular and molecular bases remain incompletely understood. Based on house dust mite (HDM)-induced mice asthma models and human samples, we applied flow cytometry, parabiosis, in vivo imaging and adoptive transplantation to confirm the persistence, migration and function of CD45⁺lineage^–CD90.2⁺NK1.1^–NKp46^–ST2^–KLRG1⁺IL-17RB⁺ memory-like ILC2s (ml-ILC2s). Regulated by CCR9/CCL25 and S1P signaling, ml-ILC2s reside in the lamina propria of small intestines (siLP) in asthma remission, and subsequently move to airway upon re-encountering antigens or alarmins. Furthermore, ml-ILC2s possess properties of longevity, potential of rapid proliferation and producing IL-13, and display transcriptional characteristics with up-regulation of Tox and Tcf-7. ml-ILC2s transplantation restore the asthmatic changes abrogated by Tox and Tcf7 knockdown. Our data identify siLP ml-ILC2s as a memory-like subset, which promotes asthma relapse. Targeting TCF-1 and TOX might be promising for preventing asthma recurrence.

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. To systematically investigate the therapeutic potential and mechanism of xanthatin ameliorating allergic airway inflammation. 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. 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). 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.

Background: Recent works imply that immune memory might be expanded to group 2 innate lymphoid cells (ILC2s), but the cellular and molecular bases are largely unknown. Here, we investigated the memory and migrating properties of Lin ^- KLRG1 ⁺ IL-17RB ⁺ ILC2s (herein referred as mILC2s) and their contribution to asthma relapse. Methods: Clinical asthmatic subjects and HDM-induced mice asthma models were applied to investigate the memory-like characteristics of mILC2s including greater effector cytokine-producing potential and in vivo persistence. Parabiosis pairs of CD45.1 ⁺ and CD45.2 ⁺ mice were employed to determine whether mILC2s were circulating cells. Adoptive transplantation was performed to analyze the origin of the mILC2s accumulated in airway upon asthma relapse. CCR9 and S1P signaling blockade were used to confirm the migration of mILC2s during different asthma phases by In vivo imaging. KLRG1 neutralization was utilized to analyze the role of mILC2s in asthma relapse on Rag1 ^-/- mice. Results: mILC2s persisted in vivo and retained the potency of producing IL-13 and re-inducing allergic responses. Critically, parabiosis study and in vivo imaging showed that the vast majority of mILC2s migrated to and resided in small intestine during asthma remission, and subsequently moved to airway upon re-encountering antigens, regulated by CCR9 and S1P signaling. Blockade of S1P signaling markedly limited secondary exposure-induced airway inflammation. Furthermore, KLRG1 neutralization attenuated asthmatic responses of Rag1 ^-/- mice, supporting a pivotal role for mILC2s in mediating asthma relapse independent of adaptive immune cells. Conclusion: mILC2s exhibit memory-like and lung-small intestine migratory properties, which empowers them to drive asthma relapse.