Detection, Isolation, and Functional Studies of Mouse Pulmonary Group 2 Innate Lymphoid Cells

ILC2s are key players in the emergence of type 2 inflammation in many pulmonary diseases. While several phenotypic markers can be used to identify ILC2s, our method utilizes the surface markers CD127 and ST2 to classify a group of type 2 cytokine-producing ILC2s upon activation by the fungal allergen Alternaria alternata . Here, we provide our protocol for the detection and isolation of a highly pure population of pulmonary mouse ILCs via flow cytometry and cell sorting. We also describe the methods for in vitro stimulation to assess the functionality of ILC2s

RNA-binding protein RBM3 intrinsically suppresses lung innate lymphoid cell activation and inflammation partially through CysLT1R.

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R