Monitoring Group 2 Innate Lymphoid Cell Biology in Models of Lung Inflammation.

Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4+ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify Lineage- Thy1.2+ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology. Key features In this protocol, ILC2s are expanded via intranasal challenges withAlternaria alternata, a fungal allergen, or recombinant IL-33. Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines. During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels ofGata-3andKi-67and produce greater amounts of IL-5 and IL-13. Graphical overview.

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.

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.

Cyclic-di-GMP Induces STING-Dependent ILC2 to ILC1 Shift During Innate Type 2 Lung Inflammation.

Type 2 inflammation is found in most forms of asthma, which may co-exist with recurrent viral infections, bacterial colonization, and host cell death. These processes drive the accumulation of intracellular cyclic-di-nucleotides such as cyclic-di-GMP (CDG). Group 2 innate lymphoid cells (ILC2s) are critical drivers of type 2 lung inflammation during fungal allergen exposure in mice; however, it is unclear how CDG regulates lung ILC responses during lung inflammation. Here, we show that intranasal CDG induced early airway type 1 interferon (IFN) production and dramatically suppressed CD127+ST2+ ILC2s and type 2 lung inflammation during Alternaria and IL-33 exposure. Further, CD127-ST2-Thy1.2+ lung ILCs, which showed a transcriptomic signature consistent with ILC1s, were expanded and activated by CDG combined with either Alternaria or IL-33. CDG-mediated suppression of type 2 inflammation occurred independent of IL-18R, IL-12, and STAT6 but required the stimulator of interferon genes (STING) and type 1 IFN signaling. Thus, CDG potently suppresses ILC2-driven lung inflammation and promotes ILC1 responses. These results suggest potential therapeutic modulation of STING to suppress type 2 inflammation and/or increase anti-viral responses during respiratory infections.