Background: Group 2 innate lymphoid cells (ILC2s) are important mediators of allergic asthma. Bacterial components such as unmethylated CpG DNA, a toll-like receptor 9 (TLR9) agonist, are known to possess beneficial immunomodulatory effects in T cell-mediated chronic asthma. However, their roles in regulating ILC2s remain unclear.

Objective: To determine the role of TLR9 activation in regulating ILC2 function and evaluate the therapeutic utility of an immunomodulatory microparticle containing natural TLR9 ligand (MIS416).

Methods: We evaluated the immunomodulatory effects of CpG A in IL-33-induced airway hyperreactivity (AHR) and airway inflammation. The roles of interferons (IFNs) were examined in vivo and in vitro using Stat1^-/- mice and neutralizing antibodies against IFN-γ and IFN-α/β receptor subunit 1 (IFNAR1), and their cellular sources were identified. The therapeutic utility of MIS416 was investigated in the Alternaria alternata model of allergic asthma and in humanized NSG mice.

Results: We show that TLR9 activation by CpG A suppresses IL-33-mediated AHR and airway inflammation through inhibition of ILC2s. Activation of TLR9 leads to production of IFN-α, which drives IFN-γ production by NK cells. Importantly, IFN-γ is essential for TLR9-driven suppression and IFN-α cannot compensate for impaired IFN-γ signalling. We further show that IFN-γ directly inhibits ILC2 function through STAT1-dependent mechanism. Finally, we demonstrate the therapeutic potential of MIS416 in A. alternata-induced airway inflammation, and validated these findings in humans.

Conclusion: TLR9 activation alleviates ILC2-driven AHR and airway inflammation through direct suppression of cell function. Microparticle-based delivery of TLR9 ligands may serve as a therapeutic strategy for asthma treatment.